Administration of a Recombinant Secretory Leukocyte Protease Inhibitor Prevents Aortic Aneurysm Growth in Mice

Administration of a Recombinant Secretory Leukocyte Protease Inhibitor Prevents Aortic Aneurysm Growth in Mice | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Administration of a Recombinant Secretory Leukocyte Protease Inhibitor Prevents Aortic Aneurysm Growth in Mice Aika Yamawaki-Ogata, Masato Mutsuga, Yuji Narita This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4239901/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 29 Aug, 2025 Read the published version in Molecular and Cellular Biochemistry → Version 1 posted 2 You are reading this latest preprint version Abstract Aim Pharmacological interventions to inhibit the progression of aortic aneurysm (AA) have not yet been established. We previously reported that mesenchymal stem cells (MSCs) provide a potential foundation for less-invasive treatment of AA. Here, we investigated secretory proteins from MSC supernatants to clarify the therapeutic effects of MSCs. Furthermore, we treated AA mice with two anti-inflammatory proteins from among these secretory proteins to confirm their therapeutic effects. Methods and Results Protein profiles of MSC-secreted factors were analyzed using protein microarrays, and two anti-inflammatory proteins, namely progranulin (PGRN) and secretory leukocyte protease inhibitor (SLPI), were identified. Apolipoprotein E-deficient mice were continuously infused with angiotensin II via osmotic pump for 4 weeks to induce AA formation, and then recombinant rPGRN and/or rSLPI were administered intraperitoneally. Mice were sacrificed at 8 weeks, and aortas were analyzed for protein expression and also stained with Elastica van Gieson and with immunofluorescence to detect macrophages. Intraperitoneal administration of rSLPI inhibited AA growth more than rPGRN alone or combined rPGRN and rSLPI, by inducing the following effects: downregulation of inflammatory cytokines and chemokines, specifically IL-1β, IL-6, TNF-α, and MCP-1; reduced of NO production; decreased phosphorylated NF-κB levels; and less of elastin destruction and macrophage infiltration. Conclusions We identified anti-inflammatory proteins, including PGRN and SLPI, in MSC supernatants, and administration of rSLPI inhibited AA progression in mice. Protein-based therapies using SLPI could be an alternative, less-invasive treatment for AA. Aortic aneurysm Mesenchymal stem cells Secretome Secretory leukocyte protease inhibitor Inflammation Macrophages Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Aortic aneurysm (AA) that dilates beyond 1.5 times the width of a normal aorta may undergo further dilation with few symptoms. While prophylactic surgical repair effectively prevents rupture, in high-risk individuals such as older, frail patients, the risks of surgical morbidity and mortality are increased [ 1 ]. Although recent endovascular aortic repair techniques effectively prevent rupture and are less invasive than previous approaches, they still have several limitations related to anatomy, migration, and endoleaks [ 2 , 3 ]. There is no established clinical pharmacological therapy to prevent AA dilation. Thus, it is necessary to develop a new, less-invasive strategy to protect against AA growth and rupture. Expanding AA is characterized by a number of proinflammatory conditions, including the following: infiltration of inflammatory cell; upregulation of inflammatory cytokines and chemokines, including interleukin (IL)-1β, IL-6, and tumor necrosis factor-α (TNF-α); increased matrix metalloproteinase (MMP) activity; and elastin degradation [ 4 ]. Currently, mesenchymal stem cell (MSC)-based therapy has been proven to be safe and effective in clinical trials in a number of fields, such as cardiology and immunology [ 5 , 6 ]. Regarding AA treatment, we previously reported that AA shrunk transiently due to the anti-inflammatory properties of intravenously administered MSCs, and we discussed the possibility of a paracrine mechanism [ 7 , 8 ]. Supernatant from MSCs is considered to be an abundant source of secretory factors, and currently serves as a cell-free therapy for various diseases. However, the MSC-derived secretome consists of diverse components with different biological effects, and it remains unclear which bioactive factors are effective against specific diseases. In this study, we investigated protein profiles of the MSC-derived secretome to identify bioactive factors (particularly anti-inflammatory factors) that may be effective for the treatment of AA. In addition, we administered recombinant proteins, including progranulin (PGRN) and secretory leukocyte protease inhibitor (SLPI), in an AA mouse model mice to determine the therapeutic effects of these proteins against AA. 2. Methods 2.1 MSC culture and protein microarray analysis Mouse bone marrow-derived MSCs were obtained using methods described in our previous studies [ 9 ]. MSC supernatant at passage 6 was collected for 48 h after starvation in fetal bovine serum (FBS)-free Dulbecco’s modified Eagle’s medium (DMEM), and cleared by centrifugation for 5 min at 1500 × g. Supernatant was concentrated 20-fold using a 3-kDa molecular weight cut-off Amicon Ultra-4 filter (Merck Millipore, Darmstadt, Germany). Supernatant without MSCs was also collected as a negative control using the same procedure. The concentration of total protein was measured using a Qubit protein assay kit on a Qubit 2.0 fluorometer (Invitrogen, Carlsbad, CA, USA). Total protein (0.4 mg) was added to a well of an array slide. The protein microarrays, which can identify 308 different mouse proteins by direct biotin labeling, were processed using L-308 Mouse Antibody Arrays (RayBiotech Life, Norcross, GA, USA). 2.2 Animals and AA models Experimental procedures followed the institutional guidelines for the care and use of laboratory animals and the ARRIVE guidelines. All experiments and procedures were approved by the Animal Experiment Advisory Committee of the Nagoya University School of Medicine (Protocol No. 20103). For this study, male apolipoprotein E deficient (apoE −/− ) mice were purchased from the Jackson Laboratory (Sacramento, CA, USA). AA was induced in forty 28-week-old male apoE −/− mice by infusion with angiotensin II (ATII; 1000 ng/kg/min, Calbiochem, Darmstadt, Germany) for 4 weeks through subcutaneous osmotic mini-pumps (model 2004; DURECT, Cupertino, CA, USA) (Daugherty et al. 2000). All mice underwent infusion and maintenance anesthesia with 1.5% isoflurane (FUJIFILM Wako Pure Chemical, Osaka, Japan). 2.3 Intraperitoneal injection of rPGRN and/or SLPI Aortic aneurysm mice which were confirmed AA formation by echography using a LOGIQ e Premium ultrasound scanner and a 10–22 MHz probe (GE Healthcare, Chicago, IL, USA) at 0 and 4 weeks after ATII infusion [ 10 ]. An AA was defined as a dilated aorta with a diameter at least 1.5 times larger at 4 weeks than that at 0 weeks, following previously published guidelines [ 11 ]. Mice with AA were divided randomly into four groups, with each of the following injected intraperitoneally: (1) 10 mg/kg rPGRN in 0.2 mL phosphate-buffered saline (PBS) (recombinant mouse, R&D Systems, Minneapolis, MN, USA; P group, n = 10), (2) 10 mg/kg rSLPI in 0.2 mL PBS (recombinant human, R&D Systems; S group, n = 10), (3) 10 mg/kg rPGRN and rSLPI in 0.2 mL PBS (PS group, n = 10), and (4) 0.2 mL saline (Saline group, n = 10). All mice underwent echography at 6 and 8 weeks. At 8 weeks, following euthanasia by an overdose of isoflurane, the aorta was carefully exposed and photographed with a DP70 digital camera (Olympus, Tokyo, Japan). 2.4 EVG staining EVG staining was performed as previously described [ 10 ]. The frozen cross-sections (10 µm) were stained for elastic lamellae using Weigert’s resorcin-fuchsin (Muto Pure Chemicals, Tokyo, Japan). Sections were photographed with a DP80 digital camera (Olympus). Images were analyzed using Cellsens software (Olympus) to determine the area of elastin staining as the percent area of elastic lamellae and the percent area of the medial component between the elastic lamellae (elastin gap area), both compared with the total medial tissue area [ 12 ]. 2.5 Immunofluorescence staining Immunofluorescence staining was performed as previously described (Ashida et al. 2023). Briefly, primary antibodies used were rat anti-inducible nitric oxide synthase (iNOS) antibody (1:50, Santa Cruz Biotechnology, Dallas, TX, USA) and rabbit anti-CD206 antibody (1:1000, Abcam, Cambridge, MA, USA). Secondary antibodies used were anti-rat IgG Alexa Fluor 488-conjugated antibody (1:5000, Cell Signaling Technology, Danvers, MA, USA) and anti-rabbit IgG Alexa Fluor 555-conjugated antibody (1:5000, Cell Signaling Technology). Negative control experiments used rat IgG1 and rabbit IgG isotype control antibodies (Cell Signaling Technology) at the same concentrations as the primary antibodies. 2.6 Enzyme-linked immunosorbent assay (ELISA) and measurement of endogenously active MMP-2 and MMP-9 ELISA assay was performed as previously described (Ashida et al. 2023). Lysate protein obtained from AA tissues was applied to each ELISA kit (IL-4, IL-10, IL-1β, IL-6, transforming growth factor (TGF)-β1, TNF-α, tissue inhibitor of metalloproteinase (TIMP)-1, and monocyte chemotactic protein (MCP)-1: Invitrogen; TIMP-2, c-Jun N-terminal kinase (JNK) 1/2 (total or phosphorylated JNK; tJNK or pJNK, pT183/Y185), nuclear factor-kappa B (NF-κB, total or phosphorylated NF-κB; tNF-κB or pNF-κB, p65), phosphorylated Smad3 (pSmad3, pS423/S425): Abcam). Endogenously active MMP-2 and MMP-9 in aortic tissues were measured using a SensoLyte 520 MMP-2 assay kit (ANASPEC, Fremont, CA, USA) and Mouse MMP-9 activity assay kit (QuickZyme Bioscience, Leiden, Netherlands). 2.7 Pretreatment of cultured macrophages with rSLPI Cell culture and expansion of murine macrophage were performed as previously described (Ashida et al. 2023). Macrophages were plated at 2× 10 4 cells per well in a 96-well plate, and pretreated with rSLPI (R&D Systems) at doses of 0, 0.1, 1, or 10 µg/mL with incubation at 37°C in a humidified atmosphere of 5% CO 2 in air for 24 h. The medium was then replaced for 24 h with growth medium containing 10 ng/mL liposaccharide (LPS, SIGMA-Aldrich) and 2 ng/mL TNF-α (recombinant human, Peprotech, Cranbury, NJ, USA). The growth medium containing LPS and TNF-α free was used as negative control. After incubation, nitric oxide (NO) production in supernatant was measured, and cells underwent RNA extraction. 2.8 Measurement of NO production To determine the volume of NO produced, supernatant was harvested from macrophages treated with rSLPI and measured using a NO2/NO3 assay kit-C II Colorimetric (Dojindo, Kumamoto, Japan). 2.9 Quantitative real-time polymerase chain reaction (qRT-PCR) Quantitative RT-PCR was performed as previously described [ 10 ]. Briefly, cDNA was synthesized using the Takara PrimeScript RT reagent kit (Takara Bio Inc., Shiga, Japan). qRT-PCR analysis was performed to determine the gene expression of IL-1β, IL-6, IL-10, iNOS, MCP-1, MMP- 2, MMP-9, NF-κB, TGF-β1, TNF-α, TIMP-1, and TIMP-2 along with β-actin (SIGMA-Aldrich) as a control (Supplemental Table 1). All data were analyzed by CFX Maestro ver.1.1 Software (Bio-Rad, Hercules, CA, USA). 2.10 Statistical analysis Statistical significance between groups was calculated by two-way repeated measure ANOVA followed by Tukey’s multiple comparisons test, Dunnet’s T3 multiple comparisons test, Dunn’s multiple comparisons test, the Mann-Whitney U test, or the Wilcoxon matched-pairs signed rank test, as appropriate, using GraphPad Prism for Mac (Version 8; GraphPad Software, San Diego, CA, USA). All error bars are expressed as standard error of the mean (SEM). Values were considered statistically different when p was < 0.05. 3. Results 3.1 Proteomic analysis of MSC supernatant Protein microarray analysis identified 256 different proteins in MSC supernatant; the details are shown in Fig. 1 A. Notably, proteins included anti-inflammatory factors such as PGRN, SLPI, IL-13, IL-27, IL-4, and TGF-β1. Figure 1 B shows that the fluorescence intensity of PGRN was highest among these, while that of SLPI was second highest. 3.2 Administration of rSLPI suppresses AA growth Aortic echography was performed according to the time course shown in Fig. 2 A. The infra-diaphragmatic to renal portion of the thoracoabdominal aorta was visualized by echography. At 4 weeks, saccular aneurysms were identified (arrow), and their maximum diameters were measured (Fig. 2 B). In all groups, the aortic diameters were significantly bigger (at least 1.5 times as large) at 4 weeks compared with 0 weeks (Fig. 2 C, p < 0.001). AA dilation progressed throughout all 8 weeks in the Saline group, whereas the P and PS groups showed no remarkable AA dilation. On the other hand, the AA diameter in the S group was significantly smaller at 6 and 8 weeks compared with 4 weeks (4 weeks: 2.20 ± 0.05 mm, 6 weeks: 1.90 ± 0.10 mm, 8 weeks: 1.92 ± 0.13 mm, 6 vs 4 weeks: p < 0.05, 8 vs 4 weeks: p < 0.05, Fig. 2 C). Microscopic findings at 8 weeks revealed that the AA was located immediately above the renal artery (Fig. 2 D). The maximum aortic short-axis diameter was significantly smaller in the S group than in the saline and P groups (S: 1.53 ± 0.17 mm, saline: 2.59 ± 0.15, P: 2.32 ± 0.22 mm, S vs Saline: p < 0.001, S vs P: p < 0.05, Fig. 2 E). 3.3 rSLPI ameliorates disruption of elastic lamellae and reduces macrophage infiltration EVG staining showed considerable degradation of the elastic lamellae in the Saline group, with thin elastin fibers in the P and PS group, whereas less disruption of the elastic lamellae was observed in the S group (Fig. 3 A). Elastic lamellae in the media were more abundant in the S group than in the Saline group (38.1 ± 1.7 vs 27 ± 2.3%, respectively, p < 0.01, Fig. 3 B). In contrast, the medial gap elastic lamellae was lower in the S group than in the Saline group (61.9 ± 1.7 vs 73 ± 2.3%, p < 0.01, Fig. 3 C). Immunofluorescence staining showed infiltration of many M1 macrophages stained with iNOS in the atherosclerosis region and media, as well as infiltration of M2 macrophages stained with CD206 in the adventitia (Fig. 4 A). The percentage of iNOS + macrophages was significantly lower in the S group than in the Saline group (11.2 ± 2.0 vs 30.9 ± 9.3%, respectively, p < 0.05, Fig. 4 B), while there was no difference in the percentage of CD206 + macrophages (Fig. 4 C). In addition, the ratio of iNOS + to CD206 + macrophages was significantly lower in the S group than in the Saline group (5.2 ± 0.9 vs 3.1 ± 0.8%, p < 0.05, Fig. 4 D). 3.4 rSLPI regulates inflammatory responses but not TIMPs or MMPs An analysis of signaling pathways showed that pNF-κB was significantly downregulated in the S group compared with the Saline group (0.4 ± 0.1 vs 1.1 ± 0.3 pg/mL, respectively, p < 0.01, Fig. 5 A), whereas there was no significant difference in the expression level of pJNK or pSmad3. Protein levels of IL-1β, IL-6, TNF-α, and MCP-1 were significantly lower in the S group than in the Saline group (IL-1β, 6.3 ± 1.1 vs 15.9 ± 2.9 pg/mL, p < 0.05; IL-6, 118.7 ± 22.2 vs 253.2 ± 23.7 pg/mL, p < 0.05; TNF-α, 41.5 ± 11.0 vs 136.6 ± 19.8 pg/mL, p < 0.01; MCP-1, 56.0 ± 3.4 vs 90.8 ± 8.6 pg/mL, p < 0.01, Fig. 5 B). Moreover, the expression levels of IL-1β, IL-6, TNF-α, and MCP-1 were significantly lower in the S group than in the P group (IL-1β, 6.3 ± 1.1 vs 17.9 ± 3.8 pg/mL, p < 0.05; IL-6, 118.7 ± 22.2 vs 192.3 ± 25.0 pg/mL, p < 0.05; TNF-α, 41.5 ± 11.0 vs 164.7 ± 25.4 pg/mL, p < 0.001; MCP-1, 56.0 ± 3.4 vs 92.1 ± 8.3 pg/mL, p < 0.01, Fig. 5 B). On the other hand, protein levels of the anti-inflammatory factors IL-4, IL-10, and TGF-β1 were significantly upregulated in the S group compared with the Saline group (IL-4, 57.8 ± 5.8 vs 24.4 ± 1.6 pg/mL, p < 0.05; IL-10, 50.6 ± 4.9 vs 33.6 ± 1.1 pg/mL, p < 0.05; TGF-β1, 432.0 ± 24.5 vs 233.6 ± 14.1 pg/mL, p < 0.001, Fig. 5 B). There were no differences between groups in the enzymatic activity levels of active MMP-2 or MMP-9 (Fig. 5 C), or in the protein levels of their respective inhibitors, TIMP-1 and TIMP-2. 3.5 rSLPI promotes downregulation of inflammatory genes and reduced NO production in cultured macrophages As shown in Fig. 6 A, we investigated the changes in mRNA expression levels in macrophages pretreated with different doses of rSLPI. Inflammatory macrophages stimulated with LPS or TNF-α promoted upregulated mRNA expression of NF-κB, IL-1β, IL-6, TNF-α, MCP-1, iNOS, and IL-10. After pretreatment by rSLPI for 24 h, macrophages exhibited no statistical differences in the mRNA expression of IL-1β, IL-6, or TNF-α, whereas the mRNA expression of NF-κB, MCP-1, and iNOS was downregulated by pretreatment with 10 µg/mL rSLPI relative to no rSLPI pretreatment (Fig. 6 A). In addition, the volume of NO2/NO3 produced by macrophages decreased dose dependently following rSLPI pretreatment (Fig. 6 B). 4. Discussion MSC-based therapy has emerged as a promising strategy in the field of regenerative medicine for various diseases, such as graft-versus-host disease, inflammatory diseases, stroke, and cardiovascular diseases [ 5 , 13 , 14 ]. Many reports have shown that MSCs produce pro- and anti-inflammatory cytokines and chemokines and regulate tissue injury responses in a transitory and paracrine manner to orchestrate tissue-repair processes [ 15 ]. However, the molecular mechanisms underlying MSC therapy are not yet well understood, so it is crucial to further investigate secretory factors released from MSCs. In this study, we profiled MSC-secreted proteins and verified the effectiveness of AA treatment using the known anti-inflammatory factors PGRN and SLPI, which are considered to be responsible for the attenuation of AA growth by MSC therapy. PGRN, which is a growth factor also referred to as granulin epithelin precursor, plays a critical role in inflammation and wound repair, and also suppresses inflammation by binding the TNF-α receptor and interrupting TNF-α signaling [ 16 ]. In several studies, administration of recombinant PGRN ameliorated renal injury, inflammatory arthritis, myocardial infarction, and acute lung injury [ 16 – 20 ]. The results in the present study showed that rPGRN was less effective than rSLPI in attenuating AA growth in mice. Importantly, while PGRN has anti-inflammatory properties, granulins cleaved from PGRN by MMP-9 and elastase have pro-inflammatory effects [ 21 ]. Progressive AA is characterized by abundant MMP-9 and neutrophil elastase in the AA wall, including the adventitia and thrombus [ 22 , 23 ]. In this study, rSLPI was administered against to existing AA in which abundant MMP-9 and neutrophil elastase were present. Therefore, it is possible that administered rPGRN underwent in enzymatic degradation by MMP-9 and neutrophil elastase. Serine proteinase inhibitors such as SLPI exert their anti-inflammatory effects by inhibiting neutrophil elastase and other leucocyte-derived serine proteinases [ 24 ]. In our in vivo experiments, intraperitoneal administration of rSLPI was most effective in inhibiting AA expansion, followed by the combination of rPGRN and rSLPI. This may be due to the fact that conversion of proinflammatory granulins from progranulin is inhibited by SLPI [ 25 ], so in mice that were administered both rPGRN and rSLPI, PGRN conversion into granulins may have been moderated by the presence of SLPI. Further results showed that rSLPI administration decreased macrophage invasion and the expression of inflammatory cytokines and chemokines, increased the expression of anti-inflammatory cytokines, and maintained elastic lamellae. These results were supported by the findings that LPS-stimulated macrophages treated with rSLPI exhibited low levels of phosphorylated NF-kB in AA tissues, reduced NO production, and downregulated expression of several mRNAs, including NF-kB, MCP-1 and iNOS. AA is characterized by strong activation of the general inflammatory transcription factor NF-kB [ 26 ]. SLPI can enter monocytes and inhibit p65 binding to the NF-kB DNA binding site [ 27 ]. In addition, LPS was shown to induce iNOS gene expression by initiating the activation of NF-KB [ 28 ]. All experiments in this study involved a single injection of one or two recombinant proteins at a concentration of 10 µg/mL after AA formation. This concentration was based on several previous studies in which rPGRN was injected intraperitoneally into diseased mice, and rSLPI was administered at the same dose [ 16 , 19 , 29 ]. Other doses, injection frequencies, and administration routes have not been studied, and further research is necessary. In conclusion, we showed that SLPI was secreted from MSC supernatant, and that administration of rSLPI inhibited AA progression in an AA mouse model. Protein-based therapies using SLPI may be beneficial as a less invasive treatment for AA, as they would carry no risk of thromboembolism associated with MSC transplants. Declarations Funding This work was supported by the Japan Society for the Promotion of Science (JSPS), KAKENHI Grant Number 22H03155. Author Contribution Statement. AYO and YN conceived and designed the study, performed data analysis, and interpreted the results. AYO collected data, performed statistical analyses, and wrote the article. YN and MM performed critical revisions of the article. YN gave final approval of the article and has overall responsibility. YN obtained funding. Acknowledgements The authors wish to acknowledge the Division for Medical Research Engineering, Nagoya University Graduate School of Medicine, for the use of a microtome cryostat and nanophotometer, and the Division for Experimental Animals, Nagoya University Graduate School of Medicine, for mouse breeding. Conflicts of Interest The authors declare no competing interests. Data Availability The data underlying this article will be shared on reasonable request to the corresponding author. References Settepani F, Cappai A, Basciu A, Barbone A, Tarelli G (2016) Outcome of open total arch replacement in the modern era. J Vasc Surg 63:537–545. 10.1016/j.jvs.2015.10.061 Schanzer A, Greenberg RK, Hevelone N, Robinson WP, Eslami MH, Goldberg RJ, Messina L (2011) Predictors of abdominal aortic aneurysm sac enlargement after endovascular repair. Circulation 123:2848–2855. 10.1161/CIRCULATIONAHA.110.014902 Schuurmann RC, Ouriel K, Muhs BE, Jordan WD Jr., Ouriel RL, Boersen JT, de Vries JP (2016) Aortic curvature as a predictor of intraoperative type Ia endoleak. J Vasc Surg 63:596–602. 10.1016/j.jvs.2015.08.110 Shimizu K, Mitchell RN, Libby P (2006) Inflammation and cellular immune responses in abdominal aortic aneurysms. Arterioscler Thromb Vasc Biol 26:987–994. 10.1161/01.ATV.0000214999.12921.4f Squillaro T, Peluso G, Galderisi U (2016) Clinical Trials With Mesenchymal Stem Cells: An Update. Cell Transpl 25:829–848. 10.3727/096368915X689622 Wu X, Jiang J, Gu Z, Zhang J, Chen Y, Liu X (2020) Mesenchymal stromal cell therapies: immunomodulatory properties and clinical progress. Stem Cell Res Ther 11:345. 10.1186/s13287-020-01855-9 Yamawaki-Ogata A, Fu X, Hashizume R, Fujimoto KL, Araki Y, Oshima H, Narita Y, Usui A (2014) Therapeutic potential of bone marrow-derived mesenchymal stem cells in formed aortic aneurysms of a mouse model. Eur J Cardiothorac Surg 45:e156–e165. 10.1093/ejcts/ezu018 Yamawaki-Ogata A, Oshima H, Usui A, Narita Y (2017) Bone marrow-derived mesenchymal stromal cells regress aortic aneurysm via the NF-kB, Smad3 and Akt signaling pathways. Cytotherapy 19:1167–1175. 10.1016/j.jcyt.2017.07.010 Hashizume R, Yamawaki-Ogata A, Ueda Y, Wagner WR, Narita Y (2011) Mesenchymal stem cells attenuate angiotensin II-induced aortic aneurysm growth in apolipoprotein E-deficient mice. J Vasc Surg 54:1743–1752. 10.1016/j.jvs.2011.06.109 Ashida S, Yamawaki-Ogata A, Tokoro M, Mutsuga M, Usui A, Narita Y (2023) Administration of anti-inflammatory M2 macrophages suppresses progression of angiotensin II-induced aortic aneurysm in mice. Sci Rep 13:1380. 10.1038/s41598-023-27412-x Johnston KW, Rutherford RB, Tilson MD, Shah DM, Hollier L, Stanley JC (1991) Suggested standards for reporting on arterial aneurysms. Subcommittee on Reporting Standards for Arterial Aneurysms, Ad Hoc Committee on Reporting Standards, Society for Vascular Surgery and North American Chapter, International Society for Cardiovascular Surgery. J Vasc Surg 13:452–458. 10.1067/mva.1991.26737 Jones JA, Beck C, Barbour JR, Zavadzkas JA, Mukherjee R, Spinale FG, Ikonomidis JS (2009) Alterations in aortic cellular constituents during thoracic aortic aneurysm development: myofibroblast-mediated vascular remodeling. Am J Pathol 175:1746–1756. 10.2353/ajpath.2009.081141 Kurtzberg J, Abdel-Azim H, Carpenter P, Chaudhury S, Horn B, Mahadeo K, Nemecek E, Neudorf S, Prasad V, Prockop S, Quigg T, Satwani P, Cheng A, Burke E, Hayes J, Skerrett D, and Group M-GS (2020) A Phase 3, Single-Arm, Prospective Study of Remestemcel-L, Ex Vivo Culture-Expanded Adult Human Mesenchymal Stromal Cells for the Treatment of Pediatric Patients Who Failed to Respond to Steroid Treatment for Acute Graft-versus-Host Disease. Biol Blood Marrow Transpl 26:845–854. 10.1016/j.bbmt.2020.01.018 Lalu MM, Mazzarello S, Zlepnig J, Dong YYR, Montroy J, McIntyre L, Devereaux PJ, Stewart DJ, David Mazer C, Barron CC, McIsaac DI, Fergusson DA (2018) Safety and Efficacy of Adult Stem Cell Therapy for Acute Myocardial Infarction and Ischemic Heart Failure (SafeCell Heart): A Systematic Review and Meta-Analysis. Stem Cells Transl Med 7:857–866. 10.1002/sctm.18-0120 Contreras-Kallens P, Terraza C, Oyarce K, Gajardo T, Campos-Mora M, Barroilhet MT, Alvarez C, Fuentes R, Figueroa F, Khoury M, Pino-Lagos K (2018) Mesenchymal stem cells and their immunosuppressive role in transplantation tolerance. Ann N Y Acad Sci 1417:35–56. 10.1111/nyas.13364 Tang W, Lu Y, Tian QY, Zhang Y, Guo FJ, Liu GY, Syed NM, Lai Y, Lin EA, Kong L, Su J, Yin F, Ding AH, Zanin-Zhorov A, Dustin ML, Tao J, Craft J, Yin Z, Feng JQ, Abramson SB, Yu XP, Liu CJ (2011) The growth factor progranulin binds to TNF receptors and is therapeutic against inflammatory arthritis in mice. Science 332:478–484. 10.1126/science.1199214 Sasaki T, Shimazawa M, Kanamori H, Yamada Y, Nishinaka A, Kuse Y, Suzuki G, Masuda T, Nakamura S, Hosokawa M, Minatoguchi S, Hara H (2020) Effects of progranulin on the pathological conditions in experimental myocardial infarction model. Sci Rep 10:11842. 10.1038/s41598-020-68804-7 Xu X, Gou L, Zhou M, Yang F, Zhao Y, Feng T, Shi P, Ghavamian A, Zhao W, Yu Y, Lu Y, Yi F, Liu G, Tang W (2016) Progranulin protects against endotoxin-induced acute kidney injury by downregulating renal cell death and inflammatory responses in mice. Int Immunopharmacol 38:409–419. 10.1016/j.intimp.2016.06.022 Zhou M, Tang W, Fu Y, Xu X, Wang Z, Lu Y, Liu F, Yang X, Wei X, Zhang Y, Liu J, Geng X, Zhang C, Wan Q, Li N, Yi F (2015) Progranulin protects against renal ischemia/reperfusion injury in mice. Kidney Int 87:918–929. 10.1038/ki.2014.403 Guo Z, Li Q, Han Y, Liang Y, Xu Z, Ren T (2012) Prevention of LPS-induced acute lung injury in mice by progranulin. Mediators Inflamm 2012:540794. 10.1155/2012/540794 De Muynck L, Van Damme P (2011) Cellular effects of progranulin in health and disease. J Mol Neurosci 45:549–560. 10.1007/s12031-011-9553-z Cohen JR, Keegan L, Sarfati I, Danna D, Ilardi C, Wise L (1991) Neutrophil chemotaxis and neutrophil elastase in the aortic wall in patients with abdominal aortic aneurysms. J Invest Surg 4:423–430. 10.3109/08941939109141172 Chen Q, Jin M, Yang F, Zhu J, Xiao Q, Zhang L (2013) Matrix metalloproteinases: inflammatory regulators of cell behaviors in vascular formation and remodeling. Mediators Inflamm 2013:928315. 10.1155/2013/928315 Hiemstra PS (2002) Novel roles of protease inhibitors in infection and inflammation. Biochem Soc Trans 30:116–120. 10.1042/ Zhu J, Nathan C, Jin W, Sim D, Ashcroft GS, Wahl SM, Lacomis L, Erdjument-Bromage H, Tempst P, Wright CD, Ding A (2002) Conversion of proepithelin to epithelins: roles of SLPI and elastase in host defense and wound repair. Cell 111:867–878. 10.1016/s0092-8674(02)01141-8 Abdul-Hussien H, Hanemaaijer R, Kleemann R, Verhaaren BF, van Bockel JH, Lindeman JH (2010) The pathophysiology of abdominal aortic aneurysm growth: corresponding and discordant inflammatory and proteolytic processes in abdominal aortic and popliteal artery aneurysms. J Vasc Surg 51:1479–1487. 10.1016/j.jvs.2010.01.057 Taggart CC, Cryan SA, Weldon S, Gibbons A, Greene CM, Kelly E, Low TB, O'Neill SJ, McElvaney NG (2005) Secretory leucoprotease inhibitor binds to NF-kappaB binding sites in monocytes and inhibits p65 binding. J Exp Med 202:1659–1668. 10.1084/jem.20050768 Griscavage JM, Wilk S, Ignarro LJ (1996) Inhibitors of the proteasome pathway interfere with induction of nitric oxide synthase in macrophages by blocking activation of transcription factor NF-kappa B. Proc Natl Acad Sci U S A 93:3308–3312. 10.1073/pnas.93.8.3308 Yu Y, Xu X, Liu L, Mao S, Feng T, Lu Y, Cheng Y, Wang H, Zhao W, Tang W (2016) Progranulin deficiency leads to severe inflammation, lung injury and cell death in a mouse model of endotoxic shock. J Cell Mol Med 20:506–517. 10.1111/jcmm.12756 Additional Declarations No competing interests reported. Supplementary Files SupplementalTable1.docx Cite Share Download PDF Status: Published Journal Publication published 29 Aug, 2025 Read the published version in Molecular and Cellular Biochemistry → Version 1 posted Submission checks completed at journal 09 Apr, 2024 First submitted to journal 09 Apr, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4239901","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":289214695,"identity":"d757d5e5-7cfc-485b-a74a-ce0d8ee5b34e","order_by":0,"name":"Aika Yamawaki-Ogata","email":"","orcid":"","institution":"Nagoya University Graduate School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Aika","middleName":"","lastName":"Yamawaki-Ogata","suffix":""},{"id":289214696,"identity":"ab56844c-0a6d-4804-a54b-83e04d4f7f08","order_by":1,"name":"Masato Mutsuga","email":"","orcid":"","institution":"Nagoya University Graduate School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Masato","middleName":"","lastName":"Mutsuga","suffix":""},{"id":289214697,"identity":"1469e281-0272-4236-8e56-16754cebda14","order_by":2,"name":"Yuji Narita","email":"data:image/png;base64,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","orcid":"","institution":"Nagoya University Graduate School of Medicine","correspondingAuthor":true,"prefix":"","firstName":"Yuji","middleName":"","lastName":"Narita","suffix":""}],"badges":[],"createdAt":"2024-04-09 05:59:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4239901/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4239901/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11010-025-05374-0","type":"published","date":"2025-08-29T15:58:15+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":54561401,"identity":"0204ce9c-2377-40d0-ab18-a38380665f43","added_by":"auto","created_at":"2024-04-12 09:59:35","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":213933,"visible":true,"origin":"","legend":"\u003cp\u003eProtein microarray analysis of MSC supernatant. (A) Two hundred fifty-six proteins were identified. Cytokines, growth factors, and chemokines were highly enriched. (B) Fluorescence intensity values of six anti-inflammatory factors: PGRN, SLPI, IL-13, IL-27, IL-4, and TGFβ-1.\u003c/p\u003e","description":"","filename":"figure.1.png","url":"https://assets-eu.researchsquare.com/files/rs-4239901/v1/adf722021c7f6bf7e7c88b53.png"},{"id":54560839,"identity":"00bf569f-5379-4e87-815c-b800a3fd5b7b","added_by":"auto","created_at":"2024-04-12 09:51:35","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":420536,"visible":true,"origin":"","legend":"\u003cp\u003eScheme of the in vivo study protocol and aortic analysis. (A) ATII was infused to apoE\u003csup\u003e-/-\u003c/sup\u003e mice for 4 weeks, then 10 mg/kg PGRN, SLPI, or saline was injected intraperitoneally (n=10 mice, respectively). Mice underwent echography at 0, 4, 6, and 8 weeks, and were sacrificed at 8 weeks. (B) Representative long-axis echography images of the thoracoabdominal aorta. Scale bars = 2 mm. (C) Aortic diameters measured by echography (n=10 mice per group). Data are means ± SEM. ***p\u0026lt;0.001 versus just before ATII infusion (week 0) within group. \u003csup\u003eθ\u003c/sup\u003ep\u0026lt;0.001 versus just before ATII infusion (week 0) within group. *p\u0026lt;0.05 versus 4 weeks in the SLPI group. \u003csup\u003e#\u003c/sup\u003ep\u0026lt;0.05 versus 4 weeks in the Saline group. \u003csup\u003e†\u003c/sup\u003ep\u0026lt;0.05 between groups at 8 weeks. Data assessed by two-way repeated measures ANOVA followed by Tukey’s multiple comparisons test. (D) Representative AA images obtained by microscopy (black arrows). Scale bars = 5 mm. (E) Aortic diameters measured by microscopy (n=10 mice per group). Data are means ± SEM. *p\u0026lt;0.05 and ***p\u0026lt;0.001 assessed by the Dunn’s multiple comparisons test. ATII: angiotensin II, PGRN: progranulin, SLPI: secretory leukocyte proteinase inhibitor.\u003c/p\u003e","description":"","filename":"figure.2.png","url":"https://assets-eu.researchsquare.com/files/rs-4239901/v1/e7b37e5f4c883c89e64709b3.png"},{"id":54560843,"identity":"61289ce2-935a-4f2b-8089-bc83b42bcbc6","added_by":"auto","created_at":"2024-04-12 09:51:36","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":323423,"visible":true,"origin":"","legend":"\u003cp\u003eQuantitative analysis of elastin in EVG-stained samples. (A) EVG staining indicates destruction of the elastic lamellar structure (black arrows). Scale bars = 200 μm Quantitative analysis of medial elastic lamellae (B), medial gap elastic lamellae (C), and number of elastic lamellae (D). Data are means ± SEM. **p\u0026lt;0.01 assessed by the Dunn’s multiple comparisons test.\u003c/p\u003e","description":"","filename":"figure.3.png","url":"https://assets-eu.researchsquare.com/files/rs-4239901/v1/1236cf534c35052f2f0f5160.png"},{"id":54561403,"identity":"d5701556-d7c4-4c70-a17c-f17d806f4d6d","added_by":"auto","created_at":"2024-04-12 09:59:36","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":546078,"visible":true,"origin":"","legend":"\u003cp\u003eQuantitative analysis of immunofluorescence images. (A) iNOS is shown in green, while CD206 is shown in red. Scale bars = 100 μm. (B) Quantitative analysis of cells positive for iNOS and CD206 (C), and the ratio of iNOS\u003csup\u003e+\u003c/sup\u003e to CD206\u003csup\u003e+\u003c/sup\u003e cells (D). Data are means ± SEM. **p\u0026lt;0.01 and ***p\u0026lt;0.001 assessed by the Dunn’s multiple comparisons test.\u003c/p\u003e","description":"","filename":"figure.4.png","url":"https://assets-eu.researchsquare.com/files/rs-4239901/v1/d2820115bef0f74f20c2aec5.png"},{"id":54560842,"identity":"1ea956ec-1961-4006-a67c-2c4ef46409d0","added_by":"auto","created_at":"2024-04-12 09:51:35","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":269401,"visible":true,"origin":"","legend":"\u003cp\u003eQuantitative analysis of protein expression levels in AA. (A) Signaling pathways of JNK, NF-kB, and smad3 (n=10 mice each). (B) ELISA analysis of inflammatory cytokines (IL-1β, IL-6, TNF-α), a chemokine (MCP-1), anti-inflammatory cytokines (IL-4, IL-10, TGF-β1), and TIMPs (TIMP-1, TIMP-2) (n=10 mice each). (C) Measurement of endogenously active MMP-2 and MMP-9 (n=10 mice each). Data are means ± SEM. *p\u0026lt;0.05, **p\u0026lt;0.01, and ***p\u0026lt;0.001 assessed by the Dunn’s multiple comparisons test.\u003c/p\u003e","description":"","filename":"figure.5.png","url":"https://assets-eu.researchsquare.com/files/rs-4239901/v1/16b5e24f19aab765b6eb0064.png"},{"id":54560846,"identity":"98bcba48-ddec-4e50-a69c-4d1c9dff376f","added_by":"auto","created_at":"2024-04-12 09:51:36","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":256338,"visible":true,"origin":"","legend":"\u003cp\u003eQuantitative analysis of mRNA expression and NO synthesis in inflammatory macrophages stimulated with SLPI. (A) Measurement of mRNA expression of NF-kB, IL-1β, IL-6, TNF-α, MCP-1, iNOS, and IL-10 (n=4 each). (B) Measurement of NO synthesis (n=4). Data are means ± SEM. *p\u0026lt;0.05, **p\u0026lt;0.01, and ***p\u0026lt;0.001 assessed by Dunnett’s T3 multiple comparisons test.\u003c/p\u003e","description":"","filename":"figure.6.png","url":"https://assets-eu.researchsquare.com/files/rs-4239901/v1/e4c8219a09621344d29691f9.png"},{"id":90344941,"identity":"2e0c3e14-0fc7-4242-a0ba-b0669c6a4b4b","added_by":"auto","created_at":"2025-09-01 16:08:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2807760,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4239901/v1/9c850b29-073f-451d-90f5-fe4674762e13.pdf"},{"id":54561991,"identity":"c75feff5-3163-4ab8-86f4-e3b7dbd23d7c","added_by":"auto","created_at":"2024-04-12 10:07:35","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":17432,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalTable1.docx","url":"https://assets-eu.researchsquare.com/files/rs-4239901/v1/6d23706a7cb07357f5e70b62.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Administration of a Recombinant Secretory Leukocyte Protease Inhibitor Prevents Aortic Aneurysm Growth in Mice","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAortic aneurysm (AA) that dilates beyond 1.5 times the width of a normal aorta may undergo further dilation with few symptoms. While prophylactic surgical repair effectively prevents rupture, in high-risk individuals such as older, frail patients, the risks of surgical morbidity and mortality are increased [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Although recent endovascular aortic repair techniques effectively prevent rupture and are less invasive than previous approaches, they still have several limitations related to anatomy, migration, and endoleaks [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. There is no established clinical pharmacological therapy to prevent AA dilation. Thus, it is necessary to develop a new, less-invasive strategy to protect against AA growth and rupture.\u003c/p\u003e \u003cp\u003eExpanding AA is characterized by a number of proinflammatory conditions, including the following: infiltration of inflammatory cell; upregulation of inflammatory cytokines and chemokines, including interleukin (IL)-1β, IL-6, and tumor necrosis factor-α (TNF-α); increased matrix metalloproteinase (MMP) activity; and elastin degradation [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Currently, mesenchymal stem cell (MSC)-based therapy has been proven to be safe and effective in clinical trials in a number of fields, such as cardiology and immunology [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Regarding AA treatment, we previously reported that AA shrunk transiently due to the anti-inflammatory properties of intravenously administered MSCs, and we discussed the possibility of a paracrine mechanism [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Supernatant from MSCs is considered to be an abundant source of secretory factors, and currently serves as a cell-free therapy for various diseases. However, the MSC-derived secretome consists of diverse components with different biological effects, and it remains unclear which bioactive factors are effective against specific diseases.\u003c/p\u003e \u003cp\u003eIn this study, we investigated protein profiles of the MSC-derived secretome to identify bioactive factors (particularly anti-inflammatory factors) that may be effective for the treatment of AA. In addition, we administered recombinant proteins, including progranulin (PGRN) and secretory leukocyte protease inhibitor (SLPI), in an AA mouse model mice to determine the therapeutic effects of these proteins against AA.\u003c/p\u003e"},{"header":"2. Methods","content":"\u003cp\u003e \u003cb\u003e2.1 MSC culture and protein microarray analysis\u003c/b\u003e \u003c/p\u003e \u003cp\u003eMouse bone marrow-derived MSCs were obtained using methods described in our previous studies [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. MSC supernatant at passage 6 was collected for 48 h after starvation in fetal bovine serum (FBS)-free Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM), and cleared by centrifugation for 5 min at 1500 \u0026times; g. Supernatant was concentrated 20-fold using a 3-kDa molecular weight cut-off Amicon Ultra-4 filter (Merck Millipore, Darmstadt, Germany). Supernatant without MSCs was also collected as a negative control using the same procedure. The concentration of total protein was measured using a Qubit protein assay kit on a Qubit 2.0 fluorometer (Invitrogen, Carlsbad, CA, USA). Total protein (0.4 mg) was added to a well of an array slide. The protein microarrays, which can identify 308 different mouse proteins by direct biotin labeling, were processed using L-308 Mouse Antibody Arrays (RayBiotech Life, Norcross, GA, USA).\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Animals and AA models\u003c/h2\u003e \u003cp\u003e Experimental procedures followed the institutional guidelines for the care and use of laboratory animals and the ARRIVE guidelines. All experiments and procedures were approved by the Animal Experiment Advisory Committee of the Nagoya University School of Medicine (Protocol No. 20103). For this study, male apolipoprotein E deficient (apoE\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e) mice were purchased from the Jackson Laboratory (Sacramento, CA, USA).\u003c/p\u003e \u003cp\u003eAA was induced in forty 28-week-old male apoE\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice by infusion with angiotensin II (ATII; 1000 ng/kg/min, Calbiochem, Darmstadt, Germany) for 4 weeks through subcutaneous osmotic mini-pumps (model 2004; DURECT, Cupertino, CA, USA) (Daugherty et al. 2000). All mice underwent infusion and maintenance anesthesia with 1.5% isoflurane (FUJIFILM Wako Pure Chemical, Osaka, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Intraperitoneal injection of rPGRN and/or SLPI\u003c/h2\u003e \u003cp\u003eAortic aneurysm mice which were confirmed AA formation by echography using a LOGIQ e Premium ultrasound scanner and a 10\u0026ndash;22 MHz probe (GE Healthcare, Chicago, IL, USA) at 0 and 4 weeks after ATII infusion [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. An AA was defined as a dilated aorta with a diameter at least 1.5 times larger at 4 weeks than that at 0 weeks, following previously published guidelines [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Mice with AA were divided randomly into four groups, with each of the following injected intraperitoneally: (1) 10 mg/kg rPGRN in 0.2 mL phosphate-buffered saline (PBS) (recombinant mouse, R\u0026amp;D Systems, Minneapolis, MN, USA; P group, \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;10), (2) 10 mg/kg rSLPI in 0.2 mL PBS (recombinant human, R\u0026amp;D Systems; S group, \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;10), (3) 10 mg/kg rPGRN and rSLPI in 0.2 mL PBS (PS group, \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;10), and (4) 0.2 mL saline (Saline group, \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;10). All mice underwent echography at 6 and 8 weeks. At 8 weeks, following euthanasia by an overdose of isoflurane, the aorta was carefully exposed and photographed with a DP70 digital camera (Olympus, Tokyo, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.4 EVG staining\u003c/h2\u003e \u003cp\u003eEVG staining was performed as previously described [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The frozen cross-sections (10 \u0026micro;m) were stained for elastic lamellae using Weigert\u0026rsquo;s resorcin-fuchsin (Muto Pure Chemicals, Tokyo, Japan). Sections were photographed with a DP80 digital camera (Olympus). Images were analyzed using Cellsens software (Olympus) to determine the area of elastin staining as the percent area of elastic lamellae and the percent area of the medial component between the elastic lamellae (elastin gap area), both compared with the total medial tissue area [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Immunofluorescence staining\u003c/h2\u003e \u003cp\u003eImmunofluorescence staining was performed as previously described (Ashida et al. 2023). Briefly, primary antibodies used were rat anti-inducible nitric oxide synthase (iNOS) antibody (1:50, Santa Cruz Biotechnology, Dallas, TX, USA) and rabbit anti-CD206 antibody (1:1000, Abcam, Cambridge, MA, USA). Secondary antibodies used were anti-rat IgG Alexa Fluor 488-conjugated antibody (1:5000, Cell Signaling Technology, Danvers, MA, USA) and anti-rabbit IgG Alexa Fluor 555-conjugated antibody (1:5000, Cell Signaling Technology). Negative control experiments used rat IgG1 and rabbit IgG isotype control antibodies (Cell Signaling Technology) at the same concentrations as the primary antibodies.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Enzyme-linked immunosorbent assay (ELISA) and measurement of endogenously active MMP-2 and MMP-9\u003c/h2\u003e \u003cp\u003eELISA assay was performed as previously described (Ashida et al. 2023). Lysate protein obtained from AA tissues was applied to each ELISA kit (IL-4, IL-10, IL-1β, IL-6, transforming growth factor (TGF)-β1, TNF-α, tissue inhibitor of metalloproteinase (TIMP)-1, and monocyte chemotactic protein (MCP)-1: Invitrogen; TIMP-2, c-Jun N-terminal kinase (JNK) 1/2 (total or phosphorylated JNK; tJNK or pJNK, pT183/Y185), nuclear factor-kappa B (NF-κB, total or phosphorylated NF-κB; tNF-κB or pNF-κB, p65), phosphorylated Smad3 (pSmad3, pS423/S425): Abcam). Endogenously active MMP-2 and MMP-9 in aortic tissues were measured using a SensoLyte 520 MMP-2 assay kit (ANASPEC, Fremont, CA, USA) and Mouse MMP-9 activity assay kit (QuickZyme Bioscience, Leiden, Netherlands).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Pretreatment of cultured macrophages with rSLPI\u003c/h2\u003e \u003cp\u003eCell culture and expansion of murine macrophage were performed as previously described (Ashida et al. 2023). Macrophages were plated at 2\u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells per well in a 96-well plate, and pretreated with rSLPI (R\u0026amp;D Systems) at doses of 0, 0.1, 1, or 10 \u0026micro;g/mL with incubation at 37\u0026deg;C in a humidified atmosphere of 5% CO\u003csub\u003e2\u003c/sub\u003e in air for 24 h. The medium was then replaced for 24 h with growth medium containing 10 ng/mL liposaccharide (LPS, SIGMA-Aldrich) and 2 ng/mL TNF-α (recombinant human, Peprotech, Cranbury, NJ, USA). The growth medium containing LPS and TNF-α free was used as negative control. After incubation, nitric oxide (NO) production in supernatant was measured, and cells underwent RNA extraction.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Measurement of NO production\u003c/h2\u003e \u003cp\u003eTo determine the volume of NO produced, supernatant was harvested from macrophages treated with rSLPI and measured using a NO2/NO3 assay kit-C II Colorimetric (Dojindo, Kumamoto, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Quantitative real-time polymerase chain reaction (qRT-PCR)\u003c/h2\u003e \u003cp\u003eQuantitative RT-PCR was performed as previously described [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Briefly, cDNA was synthesized using the Takara PrimeScript RT reagent kit (Takara Bio Inc., Shiga, Japan). qRT-PCR analysis was performed to determine the gene expression of IL-1β, IL-6, IL-10, iNOS, MCP-1, MMP- 2, MMP-9, NF-κB, TGF-β1, TNF-α, TIMP-1, and TIMP-2 along with β-actin (SIGMA-Aldrich) as a control (Supplemental Table\u0026nbsp;1). All data were analyzed by CFX Maestro ver.1.1 Software (Bio-Rad, Hercules, CA, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Statistical analysis\u003c/h2\u003e \u003cp\u003eStatistical significance between groups was calculated by two-way repeated measure ANOVA followed by Tukey\u0026rsquo;s multiple comparisons test, Dunnet\u0026rsquo;s T3 multiple comparisons test, Dunn\u0026rsquo;s multiple comparisons test, the Mann-Whitney U test, or the Wilcoxon matched-pairs signed rank test, as appropriate, using GraphPad Prism for Mac (Version 8; GraphPad Software, San Diego, CA, USA). All error bars are expressed as standard error of the mean (SEM). Values were considered statistically different when p was \u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Proteomic analysis of MSC supernatant\u003c/h2\u003e \u003cp\u003eProtein microarray analysis identified 256 different proteins in MSC supernatant; the details are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA. Notably, proteins included anti-inflammatory factors such as PGRN, SLPI, IL-13, IL-27, IL-4, and TGF-β1. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB shows that the fluorescence intensity of PGRN was highest among these, while that of SLPI was second highest.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Administration of rSLPI suppresses AA growth\u003c/h2\u003e \u003cp\u003eAortic echography was performed according to the time course shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA. The infra-diaphragmatic to renal portion of the thoracoabdominal aorta was visualized by echography. At 4 weeks, saccular aneurysms were identified (arrow), and their maximum diameters were measured (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). In all groups, the aortic diameters were significantly bigger (at least 1.5 times as large) at 4 weeks compared with 0 weeks (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). AA dilation progressed throughout all 8 weeks in the Saline group, whereas the P and PS groups showed no remarkable AA dilation. On the other hand, the AA diameter in the S group was significantly smaller at 6 and 8 weeks compared with 4 weeks (4 weeks: 2.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 mm, 6 weeks: 1.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10 mm, 8 weeks: 1.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13 mm, 6 vs 4 weeks: p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, 8 vs 4 weeks: p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Microscopic findings at 8 weeks revealed that the AA was located immediately above the renal artery (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). The maximum aortic short-axis diameter was significantly smaller in the S group than in the saline and P groups (S: 1.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17 mm, saline: 2.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15, P: 2.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22 mm, S vs Saline: p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, S vs P: p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.3 rSLPI ameliorates disruption of elastic lamellae and reduces macrophage infiltration\u003c/h2\u003e \u003cp\u003eEVG staining showed considerable degradation of the elastic lamellae in the Saline group, with thin elastin fibers in the P and PS group, whereas less disruption of the elastic lamellae was observed in the S group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Elastic lamellae in the media were more abundant in the S group than in the Saline group (38.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7 vs 27\u0026thinsp;\u0026plusmn;\u0026thinsp;2.3%, respectively, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). In contrast, the medial gap elastic lamellae was lower in the S group than in the Saline group (61.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7 vs 73\u0026thinsp;\u0026plusmn;\u0026thinsp;2.3%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eImmunofluorescence staining showed infiltration of many M1 macrophages stained with iNOS in the atherosclerosis region and media, as well as infiltration of M2 macrophages stained with CD206 in the adventitia (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). The percentage of iNOS\u003csup\u003e+\u003c/sup\u003e macrophages was significantly lower in the S group than in the Saline group (11.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0 vs 30.9\u0026thinsp;\u0026plusmn;\u0026thinsp;9.3%, respectively, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), while there was no difference in the percentage of CD206\u003csup\u003e+\u003c/sup\u003e macrophages (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). In addition, the ratio of iNOS\u003csup\u003e+\u003c/sup\u003e to CD206\u003csup\u003e+\u003c/sup\u003e macrophages was significantly lower in the S group than in the Saline group (5.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9 vs 3.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.4 rSLPI regulates inflammatory responses but not TIMPs or MMPs\u003c/h2\u003e \u003cp\u003eAn analysis of signaling pathways showed that pNF-κB was significantly downregulated in the S group compared with the Saline group (0.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 vs 1.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3 pg/mL, respectively, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA), whereas there was no significant difference in the expression level of pJNK or pSmad3. Protein levels of IL-1β, IL-6, TNF-α, and MCP-1 were significantly lower in the S group than in the Saline group (IL-1β, 6.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1 vs 15.9\u0026thinsp;\u0026plusmn;\u0026thinsp;2.9 pg/mL, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; IL-6, 118.7\u0026thinsp;\u0026plusmn;\u0026thinsp;22.2 vs 253.2\u0026thinsp;\u0026plusmn;\u0026thinsp;23.7 pg/mL, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; TNF-α, 41.5\u0026thinsp;\u0026plusmn;\u0026thinsp;11.0 vs 136.6\u0026thinsp;\u0026plusmn;\u0026thinsp;19.8 pg/mL, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; MCP-1, 56.0\u0026thinsp;\u0026plusmn;\u0026thinsp;3.4 vs 90.8\u0026thinsp;\u0026plusmn;\u0026thinsp;8.6 pg/mL, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Moreover, the expression levels of IL-1β, IL-6, TNF-α, and MCP-1 were significantly lower in the S group than in the P group (IL-1β, 6.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1 vs 17.9\u0026thinsp;\u0026plusmn;\u0026thinsp;3.8 pg/mL, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; IL-6, 118.7\u0026thinsp;\u0026plusmn;\u0026thinsp;22.2 vs 192.3\u0026thinsp;\u0026plusmn;\u0026thinsp;25.0 pg/mL, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; TNF-α, 41.5\u0026thinsp;\u0026plusmn;\u0026thinsp;11.0 vs 164.7\u0026thinsp;\u0026plusmn;\u0026thinsp;25.4 pg/mL, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; MCP-1, 56.0\u0026thinsp;\u0026plusmn;\u0026thinsp;3.4 vs 92.1\u0026thinsp;\u0026plusmn;\u0026thinsp;8.3 pg/mL, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). On the other hand, protein levels of the anti-inflammatory factors IL-4, IL-10, and TGF-β1 were significantly upregulated in the S group compared with the Saline group (IL-4, 57.8\u0026thinsp;\u0026plusmn;\u0026thinsp;5.8 vs 24.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6 pg/mL, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; IL-10, 50.6\u0026thinsp;\u0026plusmn;\u0026thinsp;4.9 vs 33.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1 pg/mL, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; TGF-β1, 432.0\u0026thinsp;\u0026plusmn;\u0026thinsp;24.5 vs 233.6\u0026thinsp;\u0026plusmn;\u0026thinsp;14.1 pg/mL, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). There were no differences between groups in the enzymatic activity levels of active MMP-2 or MMP-9 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC), or in the protein levels of their respective inhibitors, TIMP-1 and TIMP-2.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.5 rSLPI promotes downregulation of inflammatory genes and reduced NO production in cultured macrophages\u003c/h2\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, we investigated the changes in mRNA expression levels in macrophages pretreated with different doses of rSLPI. Inflammatory macrophages stimulated with LPS or TNF-α promoted upregulated mRNA expression of NF-κB, IL-1β, IL-6, TNF-α, MCP-1, iNOS, and IL-10. After pretreatment by rSLPI for 24 h, macrophages exhibited no statistical differences in the mRNA expression of IL-1β, IL-6, or TNF-α, whereas the mRNA expression of NF-κB, MCP-1, and iNOS was downregulated by pretreatment with 10 \u0026micro;g/mL rSLPI relative to no rSLPI pretreatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). In addition, the volume of NO2/NO3 produced by macrophages decreased dose dependently following rSLPI pretreatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eMSC-based therapy has emerged as a promising strategy in the field of regenerative medicine for various diseases, such as graft-versus-host disease, inflammatory diseases, stroke, and cardiovascular diseases [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Many reports have shown that MSCs produce pro- and anti-inflammatory cytokines and chemokines and regulate tissue injury responses in a transitory and paracrine manner to orchestrate tissue-repair processes [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. However, the molecular mechanisms underlying MSC therapy are not yet well understood, so it is crucial to further investigate secretory factors released from MSCs.\u003c/p\u003e \u003cp\u003eIn this study, we profiled MSC-secreted proteins and verified the effectiveness of AA treatment using the known anti-inflammatory factors PGRN and SLPI, which are considered to be responsible for the attenuation of AA growth by MSC therapy. PGRN, which is a growth factor also referred to as granulin epithelin precursor, plays a critical role in inflammation and wound repair, and also suppresses inflammation by binding the TNF-α receptor and interrupting TNF-α signaling [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In several studies, administration of recombinant PGRN ameliorated renal injury, inflammatory arthritis, myocardial infarction, and acute lung injury [\u003cspan additionalcitationids=\"CR17 CR18 CR19\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The results in the present study showed that rPGRN was less effective than rSLPI in attenuating AA growth in mice. Importantly, while PGRN has anti-inflammatory properties, granulins cleaved from PGRN by MMP-9 and elastase have pro-inflammatory effects [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Progressive AA is characterized by abundant MMP-9 and neutrophil elastase in the AA wall, including the adventitia and thrombus [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. In this study, rSLPI was administered against to existing AA in which abundant MMP-9 and neutrophil elastase were present. Therefore, it is possible that administered rPGRN underwent in enzymatic degradation by MMP-9 and neutrophil elastase.\u003c/p\u003e \u003cp\u003eSerine proteinase inhibitors such as SLPI exert their anti-inflammatory effects by inhibiting neutrophil elastase and other leucocyte-derived serine proteinases [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. In our in vivo experiments, intraperitoneal administration of rSLPI was most effective in inhibiting AA expansion, followed by the combination of rPGRN and rSLPI. This may be due to the fact that conversion of proinflammatory granulins from progranulin is inhibited by SLPI [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], so in mice that were administered both rPGRN and rSLPI, PGRN conversion into granulins may have been moderated by the presence of SLPI. Further results showed that rSLPI administration decreased macrophage invasion and the expression of inflammatory cytokines and chemokines, increased the expression of anti-inflammatory cytokines, and maintained elastic lamellae. These results were supported by the findings that LPS-stimulated macrophages treated with rSLPI exhibited low levels of phosphorylated NF-kB in AA tissues, reduced NO production, and downregulated expression of several mRNAs, including NF-kB, MCP-1 and iNOS. AA is characterized by strong activation of the general inflammatory transcription factor NF-kB [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. SLPI can enter monocytes and inhibit p65 binding to the NF-kB DNA binding site [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. In addition, LPS was shown to induce iNOS gene expression by initiating the activation of NF-KB [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAll experiments in this study involved a single injection of one or two recombinant proteins at a concentration of 10 \u0026micro;g/mL after AA formation. This concentration was based on several previous studies in which rPGRN was injected intraperitoneally into diseased mice, and rSLPI was administered at the same dose [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Other doses, injection frequencies, and administration routes have not been studied, and further research is necessary.\u003c/p\u003e \u003cp\u003eIn conclusion, we showed that SLPI was secreted from MSC supernatant, and that administration of rSLPI inhibited AA progression in an AA mouse model. Protein-based therapies using SLPI may be beneficial as a less invasive treatment for AA, as they would carry no risk of thromboembolism associated with MSC transplants.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Japan Society for the Promotion of Science (JSPS), KAKENHI Grant Number 22H03155.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contribution Statement.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAYO and YN conceived and designed the study, performed data analysis, and interpreted the results. AYO collected data, performed statistical analyses, and wrote the article. YN and MM performed critical revisions of the article. YN gave final approval of the article and has overall responsibility. YN obtained funding.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors wish to acknowledge the Division for Medical Research Engineering, Nagoya University Graduate School of Medicine, for the use of a\u0026nbsp;microtome cryostat\u0026nbsp;and nanophotometer, and the Division for Experimental Animals, Nagoya University Graduate School of Medicine, for mouse breeding.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data underlying this article will be shared on reasonable request to the corresponding author.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSettepani F, Cappai A, Basciu A, Barbone A, Tarelli G (2016) Outcome of open total arch replacement in the modern era. J Vasc Surg 63:537\u0026ndash;545. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.jvs.2015.10.061\u003c/span\u003e\u003cspan address=\"10.1016/j.jvs.2015.10.061\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchanzer A, Greenberg RK, Hevelone N, Robinson WP, Eslami MH, Goldberg RJ, Messina L (2011) Predictors of abdominal aortic aneurysm sac enlargement after endovascular repair. Circulation 123:2848\u0026ndash;2855. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1161/CIRCULATIONAHA.110.014902\u003c/span\u003e\u003cspan address=\"10.1161/CIRCULATIONAHA.110.014902\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchuurmann RC, Ouriel K, Muhs BE, Jordan WD Jr., Ouriel RL, Boersen JT, de Vries JP (2016) Aortic curvature as a predictor of intraoperative type Ia endoleak. J Vasc Surg 63:596\u0026ndash;602. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.jvs.2015.08.110\u003c/span\u003e\u003cspan address=\"10.1016/j.jvs.2015.08.110\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShimizu K, Mitchell RN, Libby P (2006) Inflammation and cellular immune responses in abdominal aortic aneurysms. Arterioscler Thromb Vasc Biol 26:987\u0026ndash;994. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1161/01.ATV.0000214999.12921.4f\u003c/span\u003e\u003cspan address=\"10.1161/01.ATV.0000214999.12921.4f\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSquillaro T, Peluso G, Galderisi U (2016) Clinical Trials With Mesenchymal Stem Cells: An Update. Cell Transpl 25:829\u0026ndash;848. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3727/096368915X689622\u003c/span\u003e\u003cspan address=\"10.3727/096368915X689622\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu X, Jiang J, Gu Z, Zhang J, Chen Y, Liu X (2020) Mesenchymal stromal cell therapies: immunomodulatory properties and clinical progress. Stem Cell Res Ther 11:345. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s13287-020-01855-9\u003c/span\u003e\u003cspan address=\"10.1186/s13287-020-01855-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYamawaki-Ogata A, Fu X, Hashizume R, Fujimoto KL, Araki Y, Oshima H, Narita Y, Usui A (2014) Therapeutic potential of bone marrow-derived mesenchymal stem cells in formed aortic aneurysms of a mouse model. Eur J Cardiothorac Surg 45:e156\u0026ndash;e165. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/ejcts/ezu018\u003c/span\u003e\u003cspan address=\"10.1093/ejcts/ezu018\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYamawaki-Ogata A, Oshima H, Usui A, Narita Y (2017) Bone marrow-derived mesenchymal stromal cells regress aortic aneurysm via the NF-kB, Smad3 and Akt signaling pathways. Cytotherapy 19:1167\u0026ndash;1175. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.jcyt.2017.07.010\u003c/span\u003e\u003cspan address=\"10.1016/j.jcyt.2017.07.010\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHashizume R, Yamawaki-Ogata A, Ueda Y, Wagner WR, Narita Y (2011) Mesenchymal stem cells attenuate angiotensin II-induced aortic aneurysm growth in apolipoprotein E-deficient mice. J Vasc Surg 54:1743\u0026ndash;1752. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.jvs.2011.06.109\u003c/span\u003e\u003cspan address=\"10.1016/j.jvs.2011.06.109\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAshida S, Yamawaki-Ogata A, Tokoro M, Mutsuga M, Usui A, Narita Y (2023) Administration of anti-inflammatory M2 macrophages suppresses progression of angiotensin II-induced aortic aneurysm in mice. Sci Rep 13:1380. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41598-023-27412-x\u003c/span\u003e\u003cspan address=\"10.1038/s41598-023-27412-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJohnston KW, Rutherford RB, Tilson MD, Shah DM, Hollier L, Stanley JC (1991) Suggested standards for reporting on arterial aneurysms. Subcommittee on Reporting Standards for Arterial Aneurysms, Ad Hoc Committee on Reporting Standards, Society for Vascular Surgery and North American Chapter, International Society for Cardiovascular Surgery. J Vasc Surg 13:452\u0026ndash;458. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1067/mva.1991.26737\u003c/span\u003e\u003cspan address=\"10.1067/mva.1991.26737\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJones JA, Beck C, Barbour JR, Zavadzkas JA, Mukherjee R, Spinale FG, Ikonomidis JS (2009) Alterations in aortic cellular constituents during thoracic aortic aneurysm development: myofibroblast-mediated vascular remodeling. Am J Pathol 175:1746\u0026ndash;1756. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.2353/ajpath.2009.081141\u003c/span\u003e\u003cspan address=\"10.2353/ajpath.2009.081141\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKurtzberg J, Abdel-Azim H, Carpenter P, Chaudhury S, Horn B, Mahadeo K, Nemecek E, Neudorf S, Prasad V, Prockop S, Quigg T, Satwani P, Cheng A, Burke E, Hayes J, Skerrett D, and Group M-GS (2020) A Phase 3, Single-Arm, Prospective Study of Remestemcel-L, Ex Vivo Culture-Expanded Adult Human Mesenchymal Stromal Cells for the Treatment of Pediatric Patients Who Failed to Respond to Steroid Treatment for Acute Graft-versus-Host Disease. Biol Blood Marrow Transpl 26:845\u0026ndash;854. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.bbmt.2020.01.018\u003c/span\u003e\u003cspan address=\"10.1016/j.bbmt.2020.01.018\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLalu MM, Mazzarello S, Zlepnig J, Dong YYR, Montroy J, McIntyre L, Devereaux PJ, Stewart DJ, David Mazer C, Barron CC, McIsaac DI, Fergusson DA (2018) Safety and Efficacy of Adult Stem Cell Therapy for Acute Myocardial Infarction and Ischemic Heart Failure (SafeCell Heart): A Systematic Review and Meta-Analysis. Stem Cells Transl Med 7:857\u0026ndash;866. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/sctm.18-0120\u003c/span\u003e\u003cspan address=\"10.1002/sctm.18-0120\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eContreras-Kallens P, Terraza C, Oyarce K, Gajardo T, Campos-Mora M, Barroilhet MT, Alvarez C, Fuentes R, Figueroa F, Khoury M, Pino-Lagos K (2018) Mesenchymal stem cells and their immunosuppressive role in transplantation tolerance. Ann N Y Acad Sci 1417:35\u0026ndash;56. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/nyas.13364\u003c/span\u003e\u003cspan address=\"10.1111/nyas.13364\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTang W, Lu Y, Tian QY, Zhang Y, Guo FJ, Liu GY, Syed NM, Lai Y, Lin EA, Kong L, Su J, Yin F, Ding AH, Zanin-Zhorov A, Dustin ML, Tao J, Craft J, Yin Z, Feng JQ, Abramson SB, Yu XP, Liu CJ (2011) The growth factor progranulin binds to TNF receptors and is therapeutic against inflammatory arthritis in mice. Science 332:478\u0026ndash;484. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1126/science.1199214\u003c/span\u003e\u003cspan address=\"10.1126/science.1199214\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSasaki T, Shimazawa M, Kanamori H, Yamada Y, Nishinaka A, Kuse Y, Suzuki G, Masuda T, Nakamura S, Hosokawa M, Minatoguchi S, Hara H (2020) Effects of progranulin on the pathological conditions in experimental myocardial infarction model. Sci Rep 10:11842. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41598-020-68804-7\u003c/span\u003e\u003cspan address=\"10.1038/s41598-020-68804-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu X, Gou L, Zhou M, Yang F, Zhao Y, Feng T, Shi P, Ghavamian A, Zhao W, Yu Y, Lu Y, Yi F, Liu G, Tang W (2016) Progranulin protects against endotoxin-induced acute kidney injury by downregulating renal cell death and inflammatory responses in mice. Int Immunopharmacol 38:409\u0026ndash;419. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.intimp.2016.06.022\u003c/span\u003e\u003cspan address=\"10.1016/j.intimp.2016.06.022\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou M, Tang W, Fu Y, Xu X, Wang Z, Lu Y, Liu F, Yang X, Wei X, Zhang Y, Liu J, Geng X, Zhang C, Wan Q, Li N, Yi F (2015) Progranulin protects against renal ischemia/reperfusion injury in mice. Kidney Int 87:918\u0026ndash;929. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/ki.2014.403\u003c/span\u003e\u003cspan address=\"10.1038/ki.2014.403\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuo Z, Li Q, Han Y, Liang Y, Xu Z, Ren T (2012) Prevention of LPS-induced acute lung injury in mice by progranulin. Mediators Inflamm 2012:540794. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1155/2012/540794\u003c/span\u003e\u003cspan address=\"10.1155/2012/540794\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDe Muynck L, Van Damme P (2011) Cellular effects of progranulin in health and disease. J Mol Neurosci 45:549\u0026ndash;560. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s12031-011-9553-z\u003c/span\u003e\u003cspan address=\"10.1007/s12031-011-9553-z\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCohen JR, Keegan L, Sarfati I, Danna D, Ilardi C, Wise L (1991) Neutrophil chemotaxis and neutrophil elastase in the aortic wall in patients with abdominal aortic aneurysms. J Invest Surg 4:423\u0026ndash;430. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3109/08941939109141172\u003c/span\u003e\u003cspan address=\"10.3109/08941939109141172\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen Q, Jin M, Yang F, Zhu J, Xiao Q, Zhang L (2013) Matrix metalloproteinases: inflammatory regulators of cell behaviors in vascular formation and remodeling. Mediators Inflamm 2013:928315. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1155/2013/928315\u003c/span\u003e\u003cspan address=\"10.1155/2013/928315\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHiemstra PS (2002) Novel roles of protease inhibitors in infection and inflammation. Biochem Soc Trans 30:116\u0026ndash;120. \u003cdiv class=\"ExternalRefDOI\"\u003e10.1042/\u003c/div\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhu J, Nathan C, Jin W, Sim D, Ashcroft GS, Wahl SM, Lacomis L, Erdjument-Bromage H, Tempst P, Wright CD, Ding A (2002) Conversion of proepithelin to epithelins: roles of SLPI and elastase in host defense and wound repair. Cell 111:867\u0026ndash;878. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/s0092-8674(02)01141-8\u003c/span\u003e\u003cspan address=\"10.1016/s0092-8674(02)01141-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAbdul-Hussien H, Hanemaaijer R, Kleemann R, Verhaaren BF, van Bockel JH, Lindeman JH (2010) The pathophysiology of abdominal aortic aneurysm growth: corresponding and discordant inflammatory and proteolytic processes in abdominal aortic and popliteal artery aneurysms. J Vasc Surg 51:1479\u0026ndash;1487. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.jvs.2010.01.057\u003c/span\u003e\u003cspan address=\"10.1016/j.jvs.2010.01.057\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTaggart CC, Cryan SA, Weldon S, Gibbons A, Greene CM, Kelly E, Low TB, O'Neill SJ, McElvaney NG (2005) Secretory leucoprotease inhibitor binds to NF-kappaB binding sites in monocytes and inhibits p65 binding. J Exp Med 202:1659\u0026ndash;1668. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1084/jem.20050768\u003c/span\u003e\u003cspan address=\"10.1084/jem.20050768\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGriscavage JM, Wilk S, Ignarro LJ (1996) Inhibitors of the proteasome pathway interfere with induction of nitric oxide synthase in macrophages by blocking activation of transcription factor NF-kappa B. Proc Natl Acad Sci U S A 93:3308\u0026ndash;3312. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1073/pnas.93.8.3308\u003c/span\u003e\u003cspan address=\"10.1073/pnas.93.8.3308\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYu Y, Xu X, Liu L, Mao S, Feng T, Lu Y, Cheng Y, Wang H, Zhao W, Tang W (2016) Progranulin deficiency leads to severe inflammation, lung injury and cell death in a mouse model of endotoxic shock. J Cell Mol Med 20:506\u0026ndash;517. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/jcmm.12756\u003c/span\u003e\u003cspan address=\"10.1111/jcmm.12756\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"molecular-and-cellular-biochemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcbi","sideBox":"Learn more about [Molecular and Cellular Biochemistry](https://www.springer.com/journal/11010)","snPcode":"11010","submissionUrl":"https://submission.nature.com/new-submission/11010/3","title":"Molecular and Cellular Biochemistry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Aortic aneurysm, Mesenchymal stem cells, Secretome, Secretory leukocyte protease inhibitor, Inflammation, Macrophages","lastPublishedDoi":"10.21203/rs.3.rs-4239901/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4239901/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eAim\u003c/h2\u003e \u003cp\u003ePharmacological interventions to inhibit the progression of aortic aneurysm (AA) have not yet been established. We previously reported that mesenchymal stem cells (MSCs) provide a potential foundation for less-invasive treatment of AA. Here, we investigated secretory proteins from MSC supernatants to clarify the therapeutic effects of MSCs. Furthermore, we treated AA mice with two anti-inflammatory proteins from among these secretory proteins to confirm their therapeutic effects.\u003c/p\u003e\u003ch2\u003eMethods and Results\u003c/h2\u003e \u003cp\u003eProtein profiles of MSC-secreted factors were analyzed using protein microarrays, and two anti-inflammatory proteins, namely progranulin (PGRN) and secretory leukocyte protease inhibitor (SLPI), were identified. Apolipoprotein E-deficient mice were continuously infused with angiotensin II via osmotic pump for 4 weeks to induce AA formation, and then recombinant rPGRN and/or rSLPI were administered intraperitoneally. Mice were sacrificed at 8 weeks, and aortas were analyzed for protein expression and also stained with Elastica van Gieson and with immunofluorescence to detect macrophages. Intraperitoneal administration of rSLPI inhibited AA growth more than rPGRN alone or combined rPGRN and rSLPI, by inducing the following effects: downregulation of inflammatory cytokines and chemokines, specifically IL-1β, IL-6, TNF-α, and MCP-1; reduced of NO production; decreased phosphorylated NF-κB levels; and less of elastin destruction and macrophage infiltration.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eWe identified anti-inflammatory proteins, including PGRN and SLPI, in MSC supernatants, and administration of rSLPI inhibited AA progression in mice. Protein-based therapies using SLPI could be an alternative, less-invasive treatment for AA.\u003c/p\u003e","manuscriptTitle":"Administration of a Recombinant Secretory Leukocyte Protease Inhibitor Prevents Aortic Aneurysm Growth in Mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-12 09:51:30","doi":"10.21203/rs.3.rs-4239901/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"checksComplete","content":"","date":"2024-04-09T10:03:55+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular and Cellular Biochemistry","date":"2024-04-09T05:46:29+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-and-cellular-biochemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcbi","sideBox":"Learn more about [Molecular and Cellular Biochemistry](https://www.springer.com/journal/11010)","snPcode":"11010","submissionUrl":"https://submission.nature.com/new-submission/11010/3","title":"Molecular and Cellular Biochemistry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"4408a6b3-f465-4dbd-b206-03223a8effeb","owner":[],"postedDate":"April 12th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-09-01T16:03:35+00:00","versionOfRecord":{"articleIdentity":"rs-4239901","link":"https://doi.org/10.1007/s11010-025-05374-0","journal":{"identity":"molecular-and-cellular-biochemistry","isVorOnly":false,"title":"Molecular and Cellular Biochemistry"},"publishedOn":"2025-08-29 15:58:15","publishedOnDateReadable":"August 29th, 2025"},"versionCreatedAt":"2024-04-12 09:51:30","video":"","vorDoi":"10.1007/s11010-025-05374-0","vorDoiUrl":"https://doi.org/10.1007/s11010-025-05374-0","workflowStages":[]},"version":"v1","identity":"rs-4239901","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4239901","identity":"rs-4239901","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}