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. 2026 May 16;46:102631. doi: 10.1016/j.bbrep.2026.102631

The role of Smoothelin-B in abdominal aortic aneurysm formation

Akiyo Fukuda a, Yutaka Koyama b, Yuya Denda a,d, Mayumi Suzuki a, Sayaka Shimodai-Yamada a, Kenta Uto a, Sander S Rensen d, Guillaume J van Eys c,1, Hiroyuki Hao a,⁎
PMCID: PMC13197721  PMID: 42180185

Abstract

Abdominal aortic aneurysm (AAA) is a life-threatening vascular disease, and its underlying mechanisms remain incompletely understood. Vascular smooth muscle cells (SMCs) are the predominant cell type in the aortic wall, and SMC dysfunction contributes to AAA development and progression. Smoothelin-B (Smtn-B) is an actin-binding protein specifically expressed in fully differentiated, contractile SMCs and is essential for maintaining the contractile apparatus. However, its role in AAA pathogenesis has yet to be clarified. To investigate this, Apoe−/− mice (single knockout, SKO) and Apoe−/− × Smtn-B−/− mice (double knockout, DKO) were subjected to an angiotensin II-induced AAA model for 28 days. AAA incidence, histopathological changes, and gene expression profiles were evaluated. AAA incidence was significantly lower in the DKO group compared with the SKO group. Histological analysis demonstrated significantly reduced Alcian blue–positive and Mac-2-positive areas in DKO mice. Gene expression levels of S100A4, F4/80, MCP-1, Ccl5, TGF-β, and Col1a1 were also significantly decreased in the DKO group. Immunohistochemical analysis further demonstrated that the S100A4-positive area was significantly reduced in the DKO group, consistent with the mRNA expression results. Smtn-B deficiency may suppress AAA development by reducing macrophage infiltration and SMC phenotypic switching via MCP-1, Ccl5, and S100A4 downregulation. These findings suggest a novel role for Smtn-B in modulating vascular inflammation and SMC phenotype during AAA pathogenesis.

Keywords: Smoothelin-B, Abdominal aortic aneurysm, Vascular smooth muscle cell

Highlights

  • •

    Smoothelin-B defines contractile smooth muscle cells and supports their function.

  • •

    Smoothelin-B loss reduced abdominal aortic aneurysm incidence in a mouse model.

  • •

    Histology and gene analysis showed reduced inflammation and macrophage infiltration.

  • •

    Smoothelin-B loss suppressed genes driving smooth muscle cell phenotype switching.

  • •

    Smoothelin-B may promote aneurysm formation by increasing inflammation and plasticity.

1. Introduction

Abdominal aortic aneurysm (AAA) development is relatively common in the elderly population, affecting approximately 5% of men ages 65 to 80 years. AAA is defined by localized dilatation (50% greater than normal aortic diameter) of the infrarenal abdominal aorta. AAA rupture is a critical consequence of this vessel dilatation and the mortality rate is high at about 80% [1]. However, the pathological mechanisms underlying AAA development have yet to be fully elucidated, and effective non-invasive therapy is lacking. Established risks for AAA are hypertension, smoking, male gender, advanced age, family history of AAA, obesity, and the presence of other large-vessel aneurysms. AAA development has been shown to involve multiple pathological processes, including phenotypic switching and dysfunction of smooth muscle cells (SMCs), genetic susceptibility, inflammation of the aortic wall, elastin degradation, oxidative stress, and extracellular matrix degradation [[2], [3], [4], [5], [6]]. Surgical replacement of the dilated portion of the aorta, including endovascular repair and open surgery, is the primary treatment for AAA. This means that patients who are poor candidates for invasive therapy face the risk of AAA rupture.

It is widely accepted that phenotypic modulation of SMCs plays an important role in various vascular diseases, such as atherosclerosis, acute coronary syndrome, AAA, aortic dissection, and so on [[7], [8], [9]]. In addition, SMCs are biologically heterogenous, and attempts, including our previous studies [7,10], have been made to isolate distinct SMC populations from arterial walls. SMCs exhibit a high degree of phenotypic plasticity, transitioning between a contractile, differentiated state and a synthetic, de-differentiated state in response to a range of stress signals [2,3]. Smoothelin-A and Smoothelin-B (Smtn-B) are present in visceral and vascular fully differentiated contractile SMCs, respectively [11,12]. Moreover, the loss of the expression of this vascular-specific subtype of smoothelin, Smtn-B, reliably reflects disappearance of the contractile SMC phenotype in various vascular pathologies, including aortic aneurysms, atherosclerosis, and restenosis [8,[13], [14], [15], [16]].

We investigated the effects of Smtn-B deletion on angiotensin II (Ang II)-induced AAA in apolipoprotein E (Apo E) knockout (Apoe−/−) mice to determine whether Smtn-B deficiency contributes to AAA formation and progression.

2. Materials and methods

2.1. Animal model and aortic tissue collection

Apoe−/− mice (single knockout; SKO) were purchased from The Jackson Laboratory, and Smtn-B−/− mice were kindly provided by Dr. Guillaume J. van Eys. Apoe−/− and Smtn-B−/− mice were interbred to generate Apoe−/− × Smtn-B−/− mice (double knockout; DKO). Genotyping of all experimental mice was confirmed by PCR using genomic DNA extracted from tail biopsies prior to inclusion in the study. Only male mice were used for this study.

All mice were maintained under specific pathogen-free conditions. Water and standard diet were provided ad libitum. All procedures involving animals were approved by the Committee at the Nihon University School of Medicine. Osmotic pumps (Alzet Osmotic Pumps, model 2004, Durect Corporation) were implanted subcutaneously at 10 weeks of age to administer Ang II at a dose of 1000 ng/kg/min for 28 days, and necropsy was performed after the 28-day infusion period. Aortic tissues (harvested from ascending, descending and abdominal portions of the aorta) were obtained from AAA and normal mice, and these aortic tissues were preserved at −80 °C or soaked in formalin and embedded in wax blocks.

2.2. Aortic diameter measurement and the incidence of AAA

In the Ang II-induced aneurysm model, the aneurysms typically develop in the suprarenal region of the aorta [17]. Prior to harvesting aortic samples, a micrometer was utilized to measure the diameter of the suprarenal portion of the aorta. Aneurysm formation was defined as a ≥50% increase in the diameter of the suprarenal portion of the aorta compared with that of the normal abdominal aorta.

2.3. Gene expression in the aortic wall

The expression levels of the following genes were analyzed using real-time PCR: SM-MHC, α-SMA, SM-calponin, S100A4, F4/80, IL-6, MCP-1, Ccl5, TGF-β and Col1a1. Total RNA was isolated from aortic tissues using the RNeasy Plus Universal Mini Kit (Qiagen, Venlo, The Netherlands) according to the manufacturer's instructions. To exclude secondary changes resulting from aneurysm formation, the aneurysmal regions were not included in the analyses. QuantiTect Reverse Transcription (Qiagen, Venlo, The Netherlands) was used to generate cDNA. This kit includes a genomic DNA elimination step using gDNA Wipeout Buffer, which was performed according to the manufacturer's instructions to minimize genomic DNA contamination prior to cDNA synthesis. TaqMan quantitative real-time PCR reactions were performed on a QuantStudio 3 Real-Time PCR System using pre-designed primer sets for SM-MHC, α-SMA, SM-calponin, S100A4, F4/80, IL-6, MCP-1, Ccl5, TGF-β, Col1a1 and the housekeeping gene GAPDH (TaqMan Gene Expression Assays, Applied Biosystems, Massachusetts, USA). mRNA expression levels were quantified by the comparative Ct method with GAPDH as the reference gene.

2.4. Histopathological analysis

Tissue sections were prepared from four regions of the harvested aorta: the distal aortic arch, thoracic aorta, aneurysmal segment or the suprarenal abdominal aorta, and the abdominal aorta (Fig. 1A and B). Tissue specimens were promptly fixed in 10% neutral-buffered formalin, embedded in paraffin, and sectioned at a thickness of 4 μm. These sections were stained with hematoxylin and eosin (HE), elastica van Gieson (EVG), Alcian blue, smooth muscle myosin heavy chain (SM-MHC), and Mac-2. HE, EVG and Alcian blue staining were performed using standard protocols. Immunohistochemical analyses were carried out using a primary rabbit polyclonal anti-SM-MHC antibody (Biomedical Technologies Inc., Madrid, Spain; dilution 1:400) and a primary rat monoclonal anti-Mac-2 antibody (clone M3/38, Cedarlane, Ontario, Canada; dilution 1:3000). To confirm the absence of Smtn-B protein expression in the DKO mice, immunohistochemical staining for Smoothelin was additionally performed using a rabbit monoclonal anti–Smoothelin antibody (clone EPR20044, Abcam, Cambridge, England; dilution 1:500). In addition, to evaluate the protein expression of molecules identified by real-time PCR analysis, immunohistochemical staining was performed using a rabbit polyclonal anti-S100A4 antibody (Agilent Technologies, California, USA; dilution 1:4000), a rabbit polyclonal anti–MCP-1 antibody (Bioss Antibodies, Massachusetts, USA; dilution 1:600), and a rabbit monoclonal anti–Ccl5/RANTES antibody (clone E9S2K, Cell Signaling Technology, Massachusetts, USA; dilution 1:200).

Fig. 1.

Fig. 1

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Macroscopic findings of the aorta and incidence of abdominal aortic aneurysm (AAA). (A) Representative macroscopic image of an abdominal aortic aneurysm in Apoe−/− mice (SKO). (B) Representative macroscopic image of the aorta from Apoe−/− × Smtn-B−/− mice (DKO) without aneurysm formation. Tissue sections were obtained from four regions: ① the distal aortic arch, ② thoracic aorta, ③ aneurysmal segment (or suprarenal abdominal aorta in mice without aneurysms), ④ abdominal aorta. (C) Incidence of AAA in SKO and DKO mice. The incidence of AAA was significantly lower in the DKO group than in the SKO group (SKO, n = 31; DKO, n = 21). (D) Aortic dilation ratio in SKO and DKO mice. The dilation ratio was calculated as the diameter of the suprarenal aorta (or the maximal short-axis diameter of the aneurysm in mice with AAA) divided by the diameter of the normal abdominal aorta. The dilation ratio was significantly lower in the DKO group compared with the SKO group (SKO, n = 30; DKO, n = 20). Each data point represents an individual mouse (biological replicate). Statistical significance was evaluated using the Kruskal–Wallis test. ∗∗∗p < 0.001.

Using ImageJ (National Institutes of Health, USA), the staining-positive areas for Alcian blue, SM-MHC, Mac-2, S100A4, MCP-1, and Ccl5 were quantified. All images were analyzed using identical image-processing settings for each staining. For Alcian blue, SM-MHC, S100A4, MCP-1, and Ccl5 staining, the positive area was normalized to the medial area of the aortic wall. In contrast, for Mac-2 staining, the positive area was normalized to the intimal plus medial area because macrophage infiltration was frequently observed in the intimal region. The ratio of each stained area to the corresponding reference area was calculated for each section, and comparisons were made between the two groups. For immunohistochemical analyses, negative staining controls were performed by omitting the primary antibody to assess nonspecific background staining.

2.5. Statistical analysis

Data were analyzed using JMP Pro 17 (SAS Institute Inc., Cary, NC, USA).

Statistical comparisons between the two groups were performed using the Kruskal–Wallis test (equivalent to the Mann–Whitney U test for two-group comparisons).

A p value < 0.05 was considered statistically significant.

3. Results

3.1. AAA incidence

The incidence of AAA was 67.7% in the SKO group (n = 31, including 5 deaths due to AAA rupture) and 9.52% in the DKO group (n = 21, including 1 death of unknown cause) (Fig. 1C). The incidence of AAA was significantly lower in the DKO than in the SKO group (p < 0.001). The dilatation ratio of the suprarenal abdominal aorta to the normal abdominal aorta was significantly lower in the DKO than in the SKO group (DKO: 1.17 ± 0.68; SKO: 2.02 ± 1.23; p < 0.0003) (Fig. 1D).

Morphologically, the aneurysms observed in the SKO mice were characterized by eccentric and irregular fusiform dilatation of the suprarenal abdominal aorta. Intraluminal thrombus formation was not observed in the examined specimens. Representative histological features of Ang II-induced AAA lesions are shown in Fig. 2.

Fig. 2.

Fig. 2

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Microscopic appearance of aortic aneurysm observed in apolipoprotein E−/− mice after 4 weeks of angiotensin II infusion. Histology of mouse aneurysm stained with hematoxylin–eosin (A) and elastica van Gieson (B). Disruption of elastic fibers in the media and dilation of the aortic wall are evident. Representative images from SKO mice with aneurysm formation are shown. Smoothelin immunostaining (C, D) demonstrates clear medial staining in SKO mice (C), whereas Smoothelin staining is absent in DKO mice (D), supporting the loss of Smtn-B expression in the aortic wall of DKO mice.

3.2. Gene expression in the aortic wall

SMCs are highly plastic cells capable of transitioning between a contractile phenotype (differentiated SMCs) and a proliferative/synthetic phenotype (dedifferentiated SMCs) [[2], [3], [4]]. Differentiated SMCs produce large amounts of contractile proteins, such as α-smooth muscle actin (α-SMA), SM-MHC, smooth muscle 22α, SM-calponin, and Smtn-B [3].

To investigate whether the loss of Smtn-B affects the expression levels of contractile smooth muscle–specific genes, we analyzed the mRNA expression levels of SM-MHC, α-SMA, and SM-calponin in the aortic vessel wall using real-time PCR. The expression levels of these representative markers of the contractile phenotype in SMCs did not differ significantly between the two groups (Fig. 3A–C). These findings are consistent with results obtained in a previous study focusing on Smtn-B [12].

Fig. 3.

Fig. 3

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Comparison of mRNA expression levels of SM-MHC (A), α-SMA (B), SM-calponin (C), S100A4 (D), F4/80 (E), IL-6 (F), MCP-1 (G), Ccl5 (H), TGF-β (I), and Col1a1 (J) between SKO mice with AAA and DKO mice without aneurysm formation. Real-time PCR shows no significant differences in the mRNA expression levels of α-SMA, SM-MHC, or SM-calponin between the two groups. The mRNA expression levels of S100A4, F4/80, MCP-1, Ccl5, TGF-β and Col1a1 were significantly lower in the DKO group, without aneurysm formation, than in the SKO group, with AAA. Each data point represents an individual mouse (biological replicate). The numbers of analyzed samples were as follows: SM-MHC (SKO n = 8, DKO n = 7), α-SMA (SKO n = 7, DKO n = 7), SM-calponin (SKO n = 8, DKO n = 7), S100A4 (SKO n = 8, DKO n = 7), F4/80 (SKO n = 6, DKO n = 7), IL-6 (SKO n = 7, DKO n = 7), MCP-1 (SKO n = 8, DKO n = 7), Ccl5 (SKO n = 7, DKO n = 7), TGF-β (SKO n = 8, DKO n = 7), and Col1a1 (SKO n = 7, DKO n = 7). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ns, not significant in Kruskal-Wallis test.

The mRNA expression level of S100A4, known to be a marker of the synthetic phenotype of SMCs [18], was also examined and found to be significantly decreased in the DKO group (Fig. 3D). Inflammatory processes within the aortic wall are critical factors in abdominal aortic remodeling [19]. The expression level of F4/80, a macrophage marker, was compared between the two groups and found to be significantly higher in the SKO group (Fig. 3E). Additionally, the mRNA expression levels of IL-6, MCP-1, Ccl5 and TGF-β were compared. The mRNA expression levels of MCP-1, Ccl5 and TGF-β were significantly lower in the DKO group (Fig. 3G–I). There was no significant difference in IL-6 expression between the two groups (Fig. 3F). Furthermore, the fibrotic marker Col1a1 was significantly upregulated in the SKO group (Fig. 3J).

3.3. Histopathological evaluation

Immunostaining for Smoothelin was performed to evaluate Smtn-B protein expression in the aortic wall. Smoothelin staining was clearly observed in the SKO group, whereas the staining intensity was markedly reduced in the DKO group, consistent with the deletion of Smtn-B at the protein level (Fig. 2C and D).

The area of acidic mucopolysaccharide deposition was evaluated by Alcian blue staining, the degree of SMC differentiation by SM-MHC staining, and the extent of macrophage infiltration by Mac-2 staining. The ratio of the acidic mucopolysaccharide deposition area to the medial area was significantly higher in the SKO than in the DKO group (Fig. 4A–C). There was no significant difference between the two groups in the ratio of the SM-MHC–positive area to the medial area (Fig. 4D–F). The ratio of the Mac-2–positive area to the intimal + medial area was significantly higher in the SKO than in the DKO group (Fig. 4G–I). The regions of interest used for quantitative analysis and an example of the threshold-based image processing are provided in Supplementary Figs. S1 and S2, respectively.

Fig. 4.

Fig. 4

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Quantitative histopathological and immunohistochemical analysis of aortic tissues. Representative images of Alcian blue staining (A, B), SM-MHC immunostaining (D, E), Mac-2 immunostaining (G, H), S100A4 immunostaining (J, K), MCP-1 immunostaining (M, N), and Ccl5 immunostaining (P, Q) in the SKO and DKO groups. Representative images from the SKO and DKO groups are shown in the upper and middle rows, respectively, and the corresponding quantitative analyses are shown in the bottom row. The corresponding quantitative analyses are shown in panels C, F, I, L, O, and R, respectively. The stained areas were quantified using ImageJ. For Alcian blue, SM-MHC, S100A4, MCP-1, and Ccl5 staining, the positive staining areas were normalized to the medial area of the aortic wall. For Mac-2 staining, the positive area was normalized to the combined intimal and medial area because macrophage infiltration was frequently observed in both layers. Each dot represents the value obtained from a single analyzed section. Sections containing advanced aneurysmal lesions were excluded to avoid secondary changes, and samples with inadequate tissue orientation were also excluded from the analysis. The numbers of analyzed sections were as follows: Alcian blue staining (SKO n = 21, DKO n = 39), SM-MHC staining (SKO n = 20, DKO n = 35), Mac-2 staining (SKO n = 29, DKO n = 37), S100A4 staining (SKO n = 15, DKO n = 15), MCP-1 staining (SKO n = 14, DKO n = 16), and Ccl5 staining (SKO n = 14, DKO n = 16). Statistical comparisons between groups were performed using the Kruskal–Wallis test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; ns, not significant.

To further evaluate the protein expression of molecules identified by real-time PCR analysis, immunohistochemical staining for S100A4, MCP-1, and Ccl5 was performed.

Quantitative analysis revealed that the S100A4-positive area normalized to the medial area was significantly reduced in the DKO group compared with the SKO group (Fig. 4J–L, p = 0.03). In contrast, MCP-1 and Ccl5 staining did not show significant differences between the two groups (Fig. 4M − O and P–R, respectively).

4. Discussion

We aimed to elucidate the pathogenesis of AAA and the functional role of Smtn-B by examining the effects of Smtn-B deletion on AAA development. Smtn-B has been recognized as a highly reliable marker of the so-called contractile phenotype of vascular SMCs, and Smtn-B deficiency reportedly reduces the contractile capacity of the aortic wall [12]. Numerous reports have described phenotypic transformation of vascular SMCs as playing essential roles in the pathogenesis of AAA [[2], [3], [4],20]. Based on these findings, we hypothesized that deficiency of Smtn-B, a marker of highly differentiated vascular SMCs, might contribute to AAA formation. However, contrary to our expectation, Smtn-B deletion resulted in a reduced incidence of AAA.

Histological analysis revealed that the areas positive for acidic mucopolysaccharide deposition and Mac-2 immunoreactivity were significantly reduced in the DKO group, suggesting that Smtn-B deficiency may suppress chronic inflammation and macrophage infiltration. Consistently, the mRNA expression level of F4/80, a macrophage marker, was significantly lower in the DKO than in the SKO group. Comparison of the expression levels of inflammation-related genes between the DKO and SKO groups revealed that IL-6 expression, which is reportedly upregulated in AAA tissues harvested from patients compared with those of controls [21], did not differ significantly between the two groups. However, the chemokines MCP-1 and Ccl5 were expressed at significantly lower levels in the DKO mice in this study than in the SKO group. Herein, to exclude secondary changes associated with aneurysm formation, mRNA was extracted from the entire aorta excluding the aneurysmal region. This approach may partly explain the lack of a significant difference in IL-6 expression between the groups of mice studied. Early stages of AAA are reportedly driven by infiltration of the vascular wall by immune cells producing inflammatory factors [22]. In the present study, the downregulation of macrophage-related gene expression, macrophage infiltration area, and MCP-1, an important chemokine known to enhance macrophage migration [23], in the DKO group suggests that Smtn-B might be involved in regulating macrophage-related gene expression and infiltration, thereby playing a role in the inflammatory response characteristic of early-phase AAA development. To further validate these transcriptional findings at the protein level, immunohistochemical analyses were performed. Smoothelin immunostaining was clearly observed in the SKO group but was markedly reduced in the DKO group, consistent with the deletion of Smtn-B at the protein level. In addition, S100A4-positive areas were significantly reduced in the DKO group compared with the SKO group, consistent with the mRNA expression results. In contrast, although MCP-1 and Ccl5 mRNA levels were decreased in the DKO mice, their protein levels assessed by immunohistochemistry did not show significant differences between the groups. This discrepancy between mRNA and protein expression may reflect post-transcriptional regulation or differences in protein stability and local tissue distribution.

Furthermore, MCP-1 and Ccl5 reportedly contribute to phenotypic switching of vascular SMCs from a contractile to a synthetic state [[23], [24], [25]]. Given that the expression levels of both genes were downregulated in the DKO group, it is reasonable to suggest that Smtn-B is involved in regulating MCP-1 and Ccl5 gene expression, and that Smtn-B deletion suppresses the expression of these genes, thereby potentially inhibiting the phenotypic switching of vascular SMCs from a contractile to a synthetic phenotype.

S100A4 is well recognized as a marker of poorly differentiated SMCs [18,26], and has also been demonstrated to play an important role in the phenotypic modulation of these cells [[26], [27], [28]]. According to Chaabane et al., treatment of classical spindle-shaped SMCs (S-SMCs) with S100A4-rich conditioned medium collected from S100A4-transfected S-SMCs induced a transition of the cells toward a poorly differentiated phenotype. Conversely, neutralization of S100A4 in poorly differentiated SMCs using an anti-S100A4 antibody promoted their transition back to the S-SMC phenotype and also decreased proliferative activity [26]. The downregulation of S100A4 gene expression observed in the DKO group suggests that Smtn-B deletion suppresses S100A4 expression, thereby promoting the transition and maintenance of vascular SMCs favoring a highly differentiated contractile phenotype.

Vascular phenotypic switching of SMCs leads to vascular dysfunction and contributes pathogenically to aneurysm formation. Smtn-B deletion may contribute to the suppression of AAA formation via the downregulation of MCP-1, Ccl5, and S100A4 gene expression, which are involved in the phenotypic switching of SMCs from a contractile to a synthetic state.

However, the present study primarily focused on the in vivo effects of Smtn-B deficiency on AAA development using a genetically modified mouse model. Therefore, the precise molecular mechanisms linking Smtn-B deficiency to the regulation of inflammatory mediators and smooth muscle cell phenotypic switching remain to be fully elucidated. Future studies using cultured vascular smooth muscle cells, including gain- and loss-of-function approaches, will be required to clarify the signaling pathways through which Smtn-B influences vascular inflammation and SMC plasticity.

TGF-β signaling is essential for maintaining both the normal structure and the functions of the aorta [29], modulating excessive activation of monocytes and macrophages [22], and preventing the progression of AAA formation [22]. In the present study, TGF-β expression was lower in the DKO group, in which AAA formation was reduced, compared with the SKO group. TGF-β signaling has been reported to be altered in AAA [19], and further studies are required to clarify the precise role of TGF-β signaling in the context of Smtn-B deficiency.

A limitation of the present study is that only male mice were used. The angiotensin II–induced AAA model in Apoe−/− mice was originally established in hyperlipidemic male mice and has been widely used for experimental AAA studies [17]. In addition, previous studies have demonstrated a marked sexual dimorphism in this model, with male mice showing a higher susceptibility to Ang II–induced AAA formation compared with female mice [30]. Future studies investigating potential sex-dependent effects of Smtn-B in AAA development would provide additional insight.

A further limitation of the present study is that a conventional global Smtn-B knockout mouse model was used. Although Smtn-B is predominantly expressed in differentiated vascular smooth muscle cells, the possible contribution of other vascular mural or mesenchymal cell populations cannot be completely excluded. Therefore, additional studies using cultured vascular smooth muscle cells and cell type–specific genetic approaches will be necessary to clarify the precise cellular mechanisms through which Smtn-B regulates AAA development.

Smtn-B deletion suppresses AAA development. In conclusion, our findings suggest that Smtn-B may be involved in promoting macrophage infiltration and the expression of macrophage-related genes. Furthermore, under conditions of vascular inflammation or atherosclerosis, Smtn-B may regulate the expression of mediators such as MCP-1, Ccl5, and S100A4, thereby modulating the phenotypic switching of vascular SMCs from a contractile to a synthetic state. Further investigation into the functions of Smtn-B may provide important insights into the pathogenesis of AAA.

Ethics statement

All animal care and experiments were conducted in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals (http://oacu.od.nih.gov/regs/index.htm. Eighth Edition; 2011) and the Basic Guidelines for Conduct of Animal Experiments published by the Ministry of Health, Labor and Welfare, Japan (https://www.mhlw.go.jp/shingi/2007/01/dl/s0124-6e.pdf). This protocol was approved by the Animal Care and Use Committee, Nihon University School of Medicine (No. AP23MED036-2).

Funding sources

This study was funded by a grant for Pioneering Research, Nihon University.

CRediT authorship contribution statement

Akiyo Fukuda: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Resources, Validation, Writing – original draft. Yutaka Koyama: Conceptualization, Data curation, Investigation, Methodology. Yuya Denda: Conceptualization, Investigation, Supervision. Mayumi Suzuki: Investigation, Project administration, Resources. Sayaka Shimodai-Yamada: Investigation, Project administration, Resources. Kenta Uto: Conceptualization, Funding acquisition, Methodology, Supervision, Validation, Writing – review & editing. Sander S. Rensen: Resources. Guillaume J. van Eys: Resources. Hiroyuki Hao: Conceptualization, Funding acquisition, Methodology, Supervision, Validation, Writing – review & editing.

Declaration of competing interest

The authors have no conflict of interest to declare.

Acknowledgements

The authors thank the staff of the Division of Human Pathology, Department of Pathology and Microbiology, and the Section of Laboratory for Animal Experiments, Institute of Medical Science, Medical Research Support Center, Nihon University School of Medicine for performing the animal experiments. We respectfully acknowledge that Dr. Guillaume van Eys, who made significant contributions to this study, passed away before the completion of this work. We honor his memory and scientific legacy.

Footnotes

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrep.2026.102631.

Contributor Information

Akiyo Fukuda, Email: akiyofukuda@hotmail.com.

Hiroyuki Hao, Email: hao.hiroyuki@nihon-u.ac.jp.

Appendix A. Supplementary data

The following are the Supplementary data to this article:

graphic file with name mmcfigs1.jpg

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Supplementary Figure S1 Regions of interest (ROI) used for quantitative histological analysis. Representative images illustrating the regions used for area quantification in each staining. The panels correspond to the representative sections shown in Fig. 4 and are labeled with the same letters followed by a prime symbol (A′, B′, D′, E′, G′, H′, J′, K′, M′, N′, P′, and Q′). Red lines indicate the manually defined regions of interest used for quantitative analysis with ImageJ. For Alcian blue, SM-MHC, S100A4, MCP-1, and Ccl5 staining, the ROI corresponds to the medial area of the aortic wall. For Mac-2 staining, the ROI includes both the intimal and medial areas because macrophage infiltration was frequently observed in both layers. Representative sections from SKO and DKO mice are shown for each staining.

graphic file with name mmcfigs2.jpg

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Supplementary Figure S2 Example of threshold-based image processing used for quantitative analysis. Representative threshold image generated during quantitative analysis using ImageJ. Image processing was performed using identical image-processing settings within each staining to identify positively stained regions. The thresholded regions correspond to the areas quantified for calculation of the positive staining area. This example corresponds to S100A4 immunostaining in the SKO group shown in Fig. 4J and Supplementary Figure S1 J′.

Data availability

Data can be obtained from the corresponding author upon reasonable request.

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