Exosomal miR-17-5p from adipose-derived mesenchymal stem cells inhibits abdominal aortic aneurysm by suppressing TXNIP-NLRP3 inflammasome

Preclinical studies have suggested that adipose-derived mesenchymal stem cells (ADSCs) transplantation can suppress abdominal aortic inflammation and aneurysm expansion through paracrine factors. Yet, the mechanism of action is not fully understood. In the present study, we further examined the function and mechanism of ADSC-derived exosomes (ADSC-exos) and their microRNA-17-5p (miR-17-5p) on the abdominal aortic aneurysm (AAA) progression. ADSC-exos were isolated and identified. DiR and PKH67 staining were used to trace ADSC-exo in vivo and in vitro. Raw264.7 cells were applied to perform in vitro experiments, while a murine AAA model induced using angiotensin II (Ang II) was used for in vivo testing. The expression level of miR-17-5p in macrophages and Ang II-treated macrophages after ADSC-exos treatment was determined using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The target relation between miR-17-5p and thioredoxin-interacting protein (TXNIP) was identified by a dual-luciferase reporter gene assay. Artificial activation and block of experiments of miR-17-5p and TXNIP were conducted to clarify their functions in inflammation during AAA progression. The severity of AAA between groups was assessed by maximal aorta diameter, AAA incidence, survival rate, and histological stainings. Besides, inflammasome-related proteins and macrophage pyroptosis were further evaluated using western blot, RT-qPCR, and enzyme-linked immunosorbent assay (ELISA). The ADSC-exos were isolated and identified. In vivo testing showed that ADSC-exos were mainly distributed in the liver. Meanwhile, in vitro experiments suggested that ADSC-derived exosomes were taken up by macrophages, while inside, ADSC-exos miR-17-5p decreased a TXNIP induced by Ang II by directly binding to its 3′-untranslated region (3’UTR). Furthermore, overexpression of miR-17-5p enhanced the therapeutic function of ADSC-exos on inflammation during AAA expansion in vivo, while its inhibition reversed this process. Finally, overexpressed TXNIP triggered macrophage pyroptosis and was alleviated by ADSC-derived exosomes in vitro. ADSC-exos miR-17-5p regulated AAA progression and inflammation via the TXNIP-NLRP3 signaling pathway, thus providing a novel insight in AAA treatment.

Background An abdominal aortic aneurysm (AAA) is a serious vascular disease characterized by segmental and permanent dilation of the abdominal aorta [1]. AAA can be dangerous if it is not spotted early on, as the rupture of an abdominal aortic aneurysm is usually lethal, with the fatality rate reaching up to 90% [2,3]. With the development of open repair and endovascular aneurysm repair (EVAR) therapy, aneurysms larger than 5.5 cm (women > 4.5 cm) in diameter are effectively treated [4,5]. However, currently, only 10% of patients are eligible for surgery, and there is no effective pharmacological treatment for patients who cannot undergo surgery [6].
AAA involves chronic inflammation of blood vessel walls. Thioredoxin-interacting protein (TXNIP) inflammasome-mediated inflammation in macrophages has an important role in various cardiovascular diseases, including AAA and atherosclerosis [7][8][9]. Usui et al. found that TXNIP-NOD-like receptor thermal protein domain associated protein 3 (NLRP3) inflammasome activation by mitochondrial oxidative stress in macrophages leads to the development of AAA, while the inhibition of NLRP3 can inhibit aneurysmal formation, arterial dilation rate, and inflammatory cell infiltration of ApoE −/− mice induced by angiotensin II (Ang II) [10]. Furthermore, Xie et al. found that transplantation of adipose-derived mesenchymal stem cells (ADSCs) can significantly reduce inflammatory cell infiltration in AAA model mice through the paracrine approach and promote the M2 macrophage polarization thus protecting against AAA [11]. Nevertheless, the proteolysis and inflammatory microenvironment of the transplanted target tissue can directly affect the survival and migration of ADSC in the transplanted site, limiting its clinical application [12].
More recently, it was found that ADSC-derived exosomes (ADSC-exos) can decrease oxidative stress, activate PI3K/Akt pathway, and increase ATP levels by enhancing myocardial viability and preventing adverse remodeling after myocardial ischemia/reperfusion injury [13,14]. Exosomes are 40-100 nm membranous microvesicles containing proteins similar to those derived from ADSCs and bioactive substances such as micro-RNAs (miRNAs), which can be exited by fusion with cell membranes and absorbed into target cells through membrane receptors or endocytosis [15]. Previous studies have discovered that exosomal miRNAs participate in the pathological process of AAA by modulating mRNAs translation [16]. For example, human mesenchymal stromal cell-derived extracellular vesicles can reduce aortic aneurysm formation and macrophage activation via miR-147 [17]. Besides, exosomal miR-106a has a pivotal role in abdominal aortic aneurysms by inducing vascular smooth muscle cell apoptosis [18]. Moreover, miR-17-5p participates in cardiovascular diseases like atherosclerosis, which is also found to decrease the thoracic aortic aneurysm [19,20]. Also, a recent study found that TXNIP is a target gene for miR-17-5p [21,22], and miR-17-5p can target TXNIP/NLRP3 pathway to suppress pancreatic β-cell pyroptosis in diabetic mice [23]. Furthermore, miR-17-5p facilitates the vascular repair of the aneurysm by regulating the PTEN-mediated PI3K/AKT/ VEGFA pathway [20]. However, the role and underlying mechanisms of exosomal miR-17-5p in AAA are not fully understood. Hence, we hypothesized that ADSC-exos could inactivate TXNIP-NLRP3 inflammasome by delivering miR-17-5p to macrophages, thus inhibiting the progression of AAA.
In this current study, our objectives were to obtain the ADSC secreted exosomes; evaluate the distribution of the ADSC-exos in vivo and in vitro; and inject the ADSCexos into the tail vein of AAA mice to demonstrate its therapeutic efficiency in vivo. Mechanistically, this study was further designed to explore the exosomal miR-17-5p of ADSCs in blocking TXNIP-NLRP3 inflammasome to acquire new strategy for the pharmaceutical treatment of AAA.

Animals
Six-week-old wild-type (WT) C57BL/6 mice and 8-weekold male ApoE −/− C57BL/6 mice were purchased from Shanghai JieSiJie Laboratory Animals Co,. LTD (Shanghai, China). All the animals were housed in an environment with a temperature of 22 ± 1 ºC, relative humidity of 50 ± 1%, and a light/dark cycle of 12/12 h. All animal studies (including the mice euthanasia procedure) were done in compliance with the regulations and guidelines of Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine institutional animal care and were conducted according to the AAALAC and the IACUC guidelines.

Dual-luciferase reporter gene assay
To analyze common binding sites between miR-17-5p and TXNIP, a dual-luciferase reporter assay was performed according to the instructions (#E1910, Promega). Raw264.7 cells were cotransfected with a luciferase reporter carrying wild-type TXNIP (TXNIP-WT) or TXNIP mutated (TXNIP-MUT) of the binding site and miR-17-5p mimics or negative control. After 6 h, the medium was replaced with complete medium. At 48 h after transfection, the luciferase activities were detected with a dual-luciferase kit (Promega). Dual-luciferase ratios (reporter genes/internal control genes) were calculated, and then, the ratio differences among different groups were compared.

Isolation and characterization of ADSC-exos
The extraction process of ADSC-exos was performed as previously described [25]. Briefly, the cell samples of ADSCs were subjected to successive centrifugations (300, 2000, and 10,000 g for 10 and 45 min, respectively), and the supernatant was collected. Subsequently, the garnered supernatant was ultracentrifuged at 110,000 × g for 75 min to obtain the precipitate that was resuspended in PBS and then subjected to centrifugation at 110,000 × g for 75 min again. Finally, the pellet was resuspended in PBS and identified by nanoparticle tracking analysis (NTA) (ZetaView, Particle Metrix, Meerbusch, Germany) to measure the diameter of the exosomes. Transmission electron microscopy (TEM) analysis (JEM-2100F, Japan Electronics, Tokyo, Japan) was used to evaluate the morphological characteristics of the exosomes. The exosome protein markers (CD63, CD81, and CD9) were determined by western blot.

SiRNA and plasmid transfection
SiRNA against TXNIP and scrambled siRNA were purchased from RiboBio, and TXNIP overexpression plasmid and control plasmid were constructed by Gene-Chem. To determine the role of TXNIP in inflammasome activation in macrophages, siRNA and plasmid were transfected into RAW264.7 cells using Lipofectamine ® 3000 (#L3000015, Thermo Fisher) according to the manufacturer's instructions. At 24 h post-transfection, the effects of gene silencing and overexpression were measured by western blot. Inflammasome activation in these cells by Ang II (10 µM, 12 h) (#A9525, Sigma) treatment was then evaluated by western blot analysis and enzymelinked immunosorbent assay [27,28].

In vivo tracing of ADSC-exos and exosomes injection
To label ADSC-exos in vivo, 1,1'-dioctadecyltetramethyl indotricarbocyanine iodide (DiR) (#22070, Beijing Fluorescence Biotechnology Co. Ltd) was dissolved in dimethylsulfoxide (DMSO) at a concentration of 1 mg/ ml. Next, DiR solution was mixed with ADSC-exos at a ratio of 2 μg DiR/100 μg ADSC-exos in PBS for 1 h and ultracentrifugated (100,000 g for 1 h) to exclude dissociative DiR and DMSO. The pellet was then resuspended in PBS at a concentration of 1 μg exosome/1μL PBS. Subsequently, PBS, DiR, and DiR-exosome were, respectively, injected via the tail vein of mice. After 72 h, in vivo fluorescence imaging machine (VISQUE In vivo Smart-LF, Vieworks, Korea) was used for fluorescence intensity analysis. ADSC-exos were then injected 100 μg per day every three days via tail vein according to the literature for a total of 28 days [29]. Specifically, we treated the Ang II-induced AAA mice with exosomes derived from ADSC vehicle , ADSC miR−17−5p inhibitor , and ADSC miR−17−5p mimics . Then AAA tissue and blood samples were collected and utilized for the following experiments.  To further validate the role of ADSC-exos in inflammasome activation, primary murine macrophages were generated from bone marrow harvested from AAA mice and cultured as previously described [30].

Cytokines detection by enzyme-linked immunosorbent assay
The cytokines concentration in cell supernatant or serum was detected using ELISA kits following the manufacturer's instructions: Mouse IL-18 ELISA kit (#ab216165, Abcam) and Mouse IL-1β ELISA kit (#KE10003, Proteintech).

Histological analysis
On day 28, mice were euthanized with the intraperitoneal injection of 0.3 ml/kg of 0.6% amobarbital and perfused with 4% paraformaldehyde (PFA) through the left cardiac apex. The entire aorta was dissected, fixed in 4% PFA, embedded in paraffin, and sectioned. The H&E, Verhoeff 's Van Gieson (EVG), Masson trichrome, and immunofluorescence stainings were performed by Shanghai Runnerbio Technology CO. Ltd (Shanghai China) according to standard procedures. For the immunofluorescence staining of the muscle sections, the F4/80 (#28463-1-AP, Proteintech) were stained with FITC (Abcam); the TXNIP (#ab188865, Abcam) was stained with cy3 (Abcam), and the nucleus was stained with DAPI (Abcam). Image J software (Rawak Software, Inc. Germany) was used for quantitative analysis of elastin degradation and immunofluorescence intensity as previously described [31].

Western blot
Total cell or tissue extracts were lysed with RIPA peptide lysis buffer containing 1% protease inhibitors (Roche; #11836153001). After being sealed with 5% bovine serum albumin (BSA) (#B2064, Sigma), the membranes were incubated with the following antibodies: anti-TXNIP, anti-NLRP3, anti-caspase-1, anti-IL-1β, anti-IL-18, and anti-GAPDH (glyceraldehyde-3phosphate dehydrogenase) at 37 °C overnight. The membranes were then rinsed three times with tris-buffered saline tween (TBST) and incubated with peroxidase-labeled secondary antibodies at room temperature for 2 h. The item number information for antibodies is listed in Table 1. Finally, the protein signals were developed using an enhanced chemiluminescence (ECL) reagent (#GERPN2106, Sigma) and visualized with a Biorad Gel Doc EQ system. The gray value was quantified using Image J software (Rawak Software, Inc. Germany).

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)
Total RNA of each sample was extracted from the cells and mouse AAA tissue using TRIzol reagent (#155960-18, Invitrogen). Then, the extracted RNA was reverse transcribed into cDNAs using a kit according to instructions (#AT341, TransGen Biotech). The cDNA template amplification was conducted by RT-qPCR using the AceQ Universal SYBR qPCR Master Mix (#Q511-03, Vazyme). A U6 transcript was used for the normalization of miR-17-5p expression; the relative mRNA expression of the genes was normalized by GAPDH. The primer sequences listed in Table 2 were synthesized by Shanghai Sangon Biotech Co., Ltd. (Shanghai, China). RT-qPCR of each sample was repeated in triplicate, and the relative expression fold change of the genes was calculated by 2 -CΔΔ t method.

Statistical analysis
GraphPad Prism version 8.0 (GraphPad, La Jolla, CA, USA, http:// www. graph pad. com) was applied for data analysis. The mean ± standard deviation was used to describe parametric values. The Chi-square test was conducted to evaluate the survival rate. The one-way analysis of variance (ANOVA) and two-tailed Student's t-test were performed to compare data between groups. A P value < 0.05 was considered statistically significant.

Isolation and characterization of ADSCs and ADSC-exos
ADSCs were collected from the inguinal region of mice and identified using flow cytometry. The ADSC-specific biomarkers such as CD29, CD31, CD34, CD45, CD90, and CD105 were positively expressed (Fig. 1A). The p3 ADSCs presented characteristics as typical spindle fibroblast-like cells (Fig. 1B). Next, positive oil-red-O or alizarin red staining of ADSCs was assessed after adipogenic differentiation induction for 2 weeks (Fig. 1C) or osteogenic differentiation induction for 3 weeks (Fig. 1D).
Western blot analysis of ADSC-exos further suggested positive expression of exosomal markers such as CD63, CD81, and CD9 (Fig. 1E). Thereafter, exosomes were generated from ADSCs supernatant by ultracentrifugation, showing a circle shape; an average particle size was 100 nm based on TEM observation and NTA (Fig. 1F,G).

Tracing of ADSC-exos in vivo and in vitro
DiR-labeled ADSC-derived exosomes were injected into mouse tail veins to track the distribution in mice ( Fig. 2A). The results showed that the fluorescence was mainly distributed in the liver (Fig. 2B), suggesting the ADSC-derived exosomes were well metabolized through the liver. The fluorescence intensity of DiR-labeled ADSC-exos was higher than that of the DiR group (P < 0.05) (Fig. 2C). Furthermore, the ex vivo influorescence images showed an obvious accumulation of the ADSC-exos signal in the liver while the signal was also observed in the kidney, lung, and spleen (Fig. 2D,E).
Next, we examined whether ADSC-exos could be taken up by Raw264.7 cells by labeling the exosomes with PKH67. Then PKH67-labeled exosomes were incubated with Raw264.7 cells (Fig. 2F). In addition, a confocal microscopy analysis showed that the labeled ADSC-derived exosomes were taken up by the macrophages (Fig. 2G), and the corresponding ratio of PKH67 positive macrophages was presented (Fig. 2H).

ADSC-exos can antagonize Ang II-induced AAA and TXNIP-NLRP3 inflammasome activation in macrophages
To determine the effect of ADSC-exos on the pathological process of AAA, we injected ADSC-exos into the tail vein of AAA mice for 28 days. The Masson trichrome staining indicated that the enhanced elastin degradation induced by Ang-II was reduced in the exosome injection group (P < 0.05) (Fig. 3A,B). Also, the elevation of serum IL-1β and IL-18 levels were alleviated by ADSC-exos treatment (P < 0.05) (Fig. 3C,D). In addition, there were fewer TXNIP positive macrophages in the area of AAA in mice treated with ADSC-exos (P < 0.05) (Fig. 3E,F). Besides, the primary murine macrophages were isolated and identified using CD68 (Fig. 3G). The upregulated level of TXNIP in primary murine macrophages was reduced by ADSC-exos (P < 0.05) (Fig. 3H-J). The data suggest that ADSC-exos can antagonize Ang II-induced AAA and TXNIP-NLRP3 inflammasome activation in macrophages.

Exosomal miR-17-5p from ADSC-derived exosomes can suppress the AAA progression
Next, we examined the role of exosomal miR-17-5p on AAA progression in vivo. Briefly, mice were randomly divided into 5 groups; each group received a different concentration of exosomal miR-17-5p in AAA (Fig. 6A). ADSC-exos treatment significantly reduced both the maximal diameter of the abdominal aorta (1.97 ± 0.36 mm) and the incidence of the AAA (73.33%) in comparison with model mice (maximal diameter of 3.56 ± 0.85 mm and AAA incidence of 93.33%. Additionally, such therapeutic effect was enhanced in the exosome miR-17-5p mimics group (maximal diameter of 1.27 ± 0.27 mm and AAA incidence of 60%, while exosomal miR-17-5p knockdown led to the opposite trend (Fig. 6B,C,E). Consistently, we also observed difference in the AAA incidence between Exo miR−17−5p inhibitor mice (86.67%) and Exo miR−17−5p NC mice. Due to the incidence of aneurysms and rupture in this model, we observed that ADSC-exos injection significantly improved the survival rate (80%) compared to that in Ang II mice (46.67%) (P = 0.0304). Among the other groups, death occurred on day 12 in the Exo miR−17−5p inhibitor group but was delayed for 5 days in the Exo miR−17−5p NC group, emphasizing that the treatment effect of the Exo miR−17−5p inhibitor group was worse than that of the Exo miR−17−5p NC group. Compared with Exo miR−17−5p NC mice, Exo miR−17−5p mimics mice died on day 18, which also presented a decreasing mortality rate (Fig. 6D).
Elastin degradation is critical for developing AAA [32]. We comprehensively evaluated the elastin degradation in the mouse AAA model by combining EVG with Masson trichrome staining (Fig. 6G). The histological analysis was consistent with the above results (P < 0.05) (Fig. 6F).
TXNIP-NLRP3 inflammasome-mediated inflammation in macrophages has an important role in various cardiovascular diseases, including AAA. Immunofluorescence analysis revealed that the TXNIP expression in F4/80 labeled macrophages in AAA of each group supported the results above (P < 0.05) (Fig. 7E,F). Then, Raw264.7 cells were treated with seven different groups of ADSC-exos, including two negative control groups, to conduct in vitro validation experiments, and the same results of the AAA mice model were reproduced (P < 0.05) (Fig. 7G-J).

ADSC-derived exosomes blocks TXNIP-mediated cell pyroptosis of macrophages in inflammation induced by Ang II
Previous studies have suggested that TXNIP regulates the activation of cell pyroptosis, thus promoting the release of cytokines like IL-18 and IL-1β [33]. In this study, the elevated expression of the positive rate of gasdermin D (GSDMD), a key molecule in the cell pyroptosis signaling, was observed in AAA tissue (Fig. 8A). Western blot analysis also elucidated that the cleaved GSDMD-N upregulated in AAA tissue (Fig. 8B,C) We have established an overexpressed TXNIP in Raw264.7 cells as mentioned above, a TXNIP siRNA plasmid was also transduced into Raw264.7 cells. Then, the mRNA level of TXNIP knockdown was validated by western blot (Fig. 8D,E) and RT-qPCR analysis (Fig. 8F). Subsequently, the Raw264.7 cells in this group were subsequently co-cultured with ADSC-exos after being treated with Ang II, and then the GSDMD-N was significantly decreased by ADSC-exos. In addition, the Raw264.7 cells in the knockdown group presented a drop in GSDMD-N. The decline of GSDMD-N was even more significant after ADSC-exos were added (Fig. 8G,H). Moreover, the elevated expression of cleaved GSDMD-N induced by Ang II was decreased by ADSC-exos, which presented an enhanced efficacy in ADSC-exos miR−17−5p mimics group. Meanwhile, miR-17-5p knockdown weakened the inhibitory effect of ADSC-exos on cleaved GSDMD-N (P < 0.05) (Fig. 8I,J). These results further identified that TXNIP triggered macrophage pyroptosis in the inflammation during AAA, which can be suppressed by exosomal miR-17-5p.

Discussion
Studies have suggested that inflammation has a critical role in the pathological process of AAA and that ADSCexos may be used to treat inflammation-related vascular diseases [34,35]. For instance, we have reported that exosomes from adipose mesenchymal stem cells overexpressing stanniocalcin-1 promoted reendothelialization after carotid endarterium mechanical injury [36]. Nevertheless, the role and underlying mechanisms of ADSC-exos in AAA progress remain poorly investigated. In the present study, we provided novel insights into the effect of ADSCderived exosomes in modulating the AAA pathological process. TXNIP exists as a ubiquitously expressed protein activated by various cellular stresses, such as inflammation and oxidative stress [37]. TXNIP can accelerate the process of releasing inflammatory cytokines. Current findings reveal that TXNIP inflammasome participates in the pathogenesis of cardiovascular diseases triggered by inflammation [38,39]. However, most investigations on the TXNIP inflammasome in AAA focus on the vascular smooth muscle cells.
Previous studies, including our research, have demonstrated the therapeutic potential of exosomes in vascular diseases, including diabetic limb ischemia, carotid endarterium injury, atherosclerosis, and abdominal aortic aneurysm [36,40,41]. In this study, we examined the role of ADSC-derived exosomes on TXNIP inflammasome inhibition. To determine the effect of ADSC-exos on the pathological process of AAA, we injected ADSC-exos into the tail vein of AAA mice for 28 days. We found that TXNIP mRNA and protein levels in macrophages were markedly reduced in AAA mice tread with ADSC-exos. Also, we observed higher cumulative survival, lower elastin degradation, and reduced inflammatory cytokines in ADSC-exos mice compared to model mice. Further experimental data suggested that this process was regulated by exosomal miR-17-5p, which can suppress AAA progression by targeting the TXNIP-NLRP3 inflammasome. Interestingly, it has been demonstrated that macrophage-derived exosomes trigger MMP-2 expression in VSMC via JNK and p38 pathways, indicating another pathophysiological role of exosomes involved in the pathogenesis of AAA. In this present study, we found ADSC-exos containing different levels of miR-17-5p presented different therapeutic effects [35]. Specifically, the difference in the AAA incidence and between Exo miR−17−5p inhibitor mice and Exo miR−17−5p NC mice were observed, indicating exosomal miR-17-5p knockdown weakened the therapeutic efficacy of ADSC-exos. It indicated that the miR-17-5p-TXNIP axon played one of the essential roles in exosomes treatment for AAA. Taken together, these data provided the first functional evidence that miR-17-5p from ADSC-exos pivotally modulates TXNIP inflammasome activation in macrophages, suggesting exosomal miR-17-5p injection may become a future target for constraining the progression of AAA. What is noteworthy, miR-17-5p has been demonstrated to alleviates myocardial ischemia-reperfusion injury by regulating cell pyroptosis, which was a new paradigm of cell death for fighting against vascular diseases [42][43][44].
Cell pyroptosis is a caspase-1-dependent proinflammatory cell death characterized as cell swelling and dissolution of the cell membrane, which can be induced by triggering TXNIP-NLRP3 signaling [45]. This process is accompanied by the release of many inflammatory factors such as IL-1β and IL-18, which ultimately magnifies the local inflammatory response. Xiong et al. found that regulation of vascular smooth muscle cell pyroptosis could constrain AAA [46]. Moreover, macrophage pyroptosis has been suggested to be involved in AAA [47]. Our study elucidated that Ang II activated GSDMD in the macrophages of AAA mice, which was upregulated or downregulated by TXNIP artificial overexpression or knockdown in vitro. Moreover, the decline of GSDMD-N in the knockdown group was even more significant after ADSC-exos were added, while the upregulation of GSDMD was offset by ADSC-exos treatment. To sum up, our findings suggested that ADSC-exos might be a novel therapeutic approach for prevention of detrimental influences brought about by macrophages pyroptosis during AAA expansion (Fig. 8K). Taken together, we inferred that miR-17-5p carried by ADSC-derived exosomes alleviated AAA progression by inhibiting the TXNIP-NLRP3 inflammasome signaling pathway. However, this study has some limitations. First, although the murine AAA model of Ang II infusion in ApoE −/− C57BL/6 mice simultaneously receiving HFD for 28 days has been reported to mimic hypertension and hyperlipidemia of clinical AAA patients [48], there are other risk factors such as elastin degradation in AAA pathophysiological changes. Thereafter, further efforts should be made to improve the modeling method in order to better conform to the clinical situation of AAA. In addition, we artificially constructed activated or inactivated TXNIP at the cellular level to conduct a preliminary investigation of the underlying mechanisms in this project. Given the complex environment in vivo during the development of AAA, further TXNIP interference in AAA mice should be performed to confirm our deduction. Studies on TXNIP interference in vivo were carried out by our research team, which may help verify the functions and mechanisms of ADSC-derived exosomes in restraining AAA expansion via the TXNIP-NLRP3 signaling pathway.

Conclusion
This study demonstrated the significant role of miR-17-5p from ADSC-exos in constraining AAA progression, inflammatory cytokines release, and TXNIP-NLRP3 inflammasome in vivo. In vitro further revealed that miR-17-5p could reduce macrophage pyroptosis during inflammation induced by Ang II by targeting TXNIP. Hence, miR-17-5p-rich ADSC-exos may become a new strategy in the clinical practice for using ADSC-derived exosomes in AAA treatment.