Abstract

   Abdominal aortic aneurysm (AAA) is a high‐risk inflammatory disorder.
   SENP3, a SUMO2/3‐specific protease, is closely involved in the
   development of cancer. In this study, the aim is to explore the role of
   SENP3 in macrophages in AAA. It is found that the protein expression of
   SENP3 is significantly upregulated in both human and murine AAA
   specimens. SENP3 expression is negatively regulated by the E3 ubiquitin
   ligase STUB1/CHIP. Furthermore, myeloid‐specific SENP3 knockout
   inhibited AAA formation in both AngII‐ and CaCl[2]‐induced mouse
   models. SENP3 deficiency repressed AAA lesion macrophage infiltration
   and inflammatory response. Mechanistic studies identified Cystathionine
   Gamma–Lyase (CTH), a critical enzyme involved in hydrogen sulfide
   production, as a target protein of SENP3 that mediated the exacerbating
   effects of SENP3 on ferroptosis and inflammatory programs in
   macrophages. SUMO‐3 modification at Lysine 361 promoted CTH protein
   stability, whereas de‐SUMOylation by SENP3 facilitated its
   proteasome‐dependent degradation. Most importantly, it is found that
   CTH inhibitor counteracted the protective effect of SENP3 deficiency on
   AAA. Additionally, supplementation with ATB346, a novel H[2]S‐donating
   naproxen derivative, prevented AAA development in mice. These studies
   suggest that SENP3‐mediated CTH deSUMOylation regulates macrophage
   ferroptosis and AAA development. The SENP3/CTH axis is therefore an
   important therapeutic target for aortic aneurysmal diseases.

   Keywords: abdominal aortic aneurysm, CTH, ferroptosis, SENP3,
   SUMOylation
     __________________________________________________________________

   In this study, it is proposed that SENP3 accumulates in macrophages of
   aneurysmal aortic tissues, which promotes macrophage ferroptosis and
   inflammation via regulation of CTH de‐SUMOylation.

   graphic file with name ADVS-12-2414500-g008.jpg

1. Introduction

   Abdominal aortic aneurysm (AAA) is an age‐related disease,
   characterized by progressive segmental dilation of the abdominal aorta
   of 50% or more compared to the normal diameter, with an ≈80% mortality
   rate upon rupture.^[ [54]^1 , [55]^2 ^] Currently, endovascular
   stenting and open surgery are the mainstays of treatment for AAA, and
   no medication has been approved by the U.S. Food and Drug
   Administration (FDA) to induce regression or slow AAA growth and
   restrain the risk of rupture.^[ [56]^3 ^] Thus, clarifying the
   potential molecular mechanisms and mining novel and efficient
   therapeutic targets are crucial.

   Inflammation is the hallmark of AAA with macrophages being predominant.
   In clinical samples and animal models, macrophages play an essential
   and distinct role in AAA development and progression to rupture.^[
   [57]^4 ^] During vascular remodeling, monocytes are recruited to
   aneurysm tissues and differentiate into macrophages that participate in
   inflammatory cytokine expression, proteolytic enzyme production,
   extracellular matrix degradation, and pathological aortic dilation,
   together with specialized tissue‐resident macrophages.^[ [58]^5 ^]
   However, the critical pathways through which macrophages facilitate
   inflammation during AAA formation remain insufficiently elucidated.
   Ferroptosis is an iron‐dependent form of cell death, which is
   characterized by the production of reactive oxygen species (ROS) from
   accumulated iron and lipid peroxidation.^[ [59]^6 ^] Previous studies
   have indicated that ferroptosis facilitates the progression and
   associated inflammation of AAA.^[ [60]^7 , [61]^8 , [62]^9 ^]
   Macrophage ferroptosis is therefore a potential therapeutic target for
   prevention of AAA development.

   Post‐translational modifications (PTMs), such as ubiquitination,
   phosphorylation, glycosylation, and SUMOylation, play important roles
   in modulating protein functions. Among them, SUMOylation entails the
   conjugation of Small Ubiquitin‐like Modifier (SUMO) to lysine
   residue(s) on target proteins, thereby influencing the subcellular
   localization, protein stability, and protein‐protein interactions.
   Notably, SUMOylation can be reversed by Sentrin/SUMO‐specific proteases
   (SENPs), releasing the SUMO molecule(s) for the next cycle.^[ [63]^10
   ^] We have previously demonstrated that SENP3, one of the members of
   SENPs family, plays critical roles in cardiovascular and metabolic
   diseases via its de‐SUMOylation of protein substrates.^[ [64]^11 ,
   [65]^12 ^] SENP3 promotes vascular smooth muscle cell (VSMC)
   proliferation and migration in vascular remodeling via de‐SUMOylation
   of β‐catenin.^[ [66]^11 ^] SENP3 de‐SUMOylates YAP1 and regulates
   macrophage inflammation during diet and age‑induced obesity.^[ [67]^12
   ^] However, the role of SENP3 in aortic aneurysmal diseases has never
   been investigated.

   In this study, we aimed to investigate whether SENP3 mediates the
   pathophysiology of AAA, and to explore possible underlying molecular
   mechanisms. We found that SENP3 contributes to AAA formation and
   macrophage ferroptosis by targeting cystathionine‐γ‐lyase (CTH), a
   hydrogen sulfide (H[2]S)‐synthesizing enzyme. Moreover, ATB346, a novel
   H[2]S‐releasing drug, ameliorates the vascular inflammation and
   restricted AAA formation.

2. Result

2.1. The Protein Expression of SENP3 is Markedly Upregulated in Human and
Murine AAA Tissues

   To explore whether SENP3 is involved in the pathogenesis of AAA, we
   first examined the expression of SENP3 in AAA tissues. SENP3 protein
   levels were strongly increased, whereas the mRNA levels remained
   unaltered in human AAA tissues (Figure [68] 1A–C). Consistently, SENP3
   protein levels were markedly increased in the aneurysmal aortas from
   AngII‐infused ApoE^−/− mice (Figure [69]1D–F) and CaCl[2]‐treated
   C57BL/6J mice (Figure [70]S1A,B, Supporting Information).
   Immunofluorescence staining revealed a heightened presence of
   infiltrating macrophages in both human and murine AAA tissues, with
   high expression of SENP3 within these macrophages (Figure [71]1G,H;
   Figure [72]S1C, Supporting Information).

Figure 1.

   Figure 1
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   The protein expression of SENP3 is significantly upregulated in human
   and murine AAA tissues. A–C) SENP3 protein and mRNA levels were
   determined by western blot (A,B) and real‐time quantitative polymerase
   chain reaction (RT‐qPCR; C) in human abdominal aortic aneurysm (AAA)
   samples and adjacent nonaneurysmal control samples (non‐AAA) (n = 6 per
   group). D–F) SENP3 protein and mRNA levels were determined by western
   blot (D,E) and RT‐qPCR (F) in abdominal aortic samples of ApoE^−/− mice
   infused with angiotensin II (AngII) or saline for 28 days (n = 6 per
   group). G) Representative images of dual immunofluorescence staining of
   SENP3 (green) and CD68 (red) in human AAA samples and adjacent
   nonaneurysmal control samples. Where indicated, nuclei were
   counterstained with 4′,6‐diamidino‐2‐phenylindole (DAPI; blue). Scale
   bar: 100 µm. H) Representative images of dual immunofluorescence
   staining for SENP3 (green) and CD68 (red) in abdominal aortic samples
   of ApoE^−/− mice infused with AngII or saline for 28 days. Scale bar:
   100 µm. I,J) SENP3 protein levels were determined by western blot in
   BMDMs after stimulation with AngII (1 µmol L^−1) for the indicated time
   periods (n = 4 per group). K,L) SENP3 protein levels were determined by
   western blot in bone marrow‐derived macrophages (BMDMs) after
   stimulation with indicated concentrations of AngII for 180 min (n = 4
   per group). M,N) Representative images of dual immunofluorescence
   staining for SENP3 (green) and CD68 (red) in BMDMs stimulated with
   AngII (1 µmol L^−1) for 180 min (M). Where indicated, nuclei were
   counterstained with DAPI (blue). Scale bar: 25 µm. Quantification of
   fluorescence intensity of SENP3 (N; n = 4 for each group). Data
   represent mean ± SEM. p values were determined using the student's
   t‐test (B,C,E,F,N) and Welch ANOVA test (J,L). For all panels, *p <
   0.05; **p < 0.01; ***p < 0.001; ns: not significant (p > 0.05).

   To further investigate the role of SENP3 in macrophages in AAA, mouse
   bone marrow‐derived macrophages (BMDMs) and human THP‐1‐derived
   macrophages were treated with AngII in vitro. AngII markedly increased
   SENP3 protein expression in a time‐ and dose‐dependent manner
   (Figure [74]1I–L; Figure [75]S2, Supporting Information). However, no
   significant change in SENP3 mRNA level was observed after AngII
   stimulation (Figure [76]S3A,B, Supporting Information).
   Immunofluorescence staining further confirmed the upregulation of SENP3
   protein levels in BMDMs after AngII stimulation (Figure [77]1M,N).
   Moreover, we found that AngII increased intracellular ROS levels in
   BMDMs and triggered SENP3 accumulation, which was completely abolished
   by ROS scavengers phenyl N‐t‐butylnitrone (PBN) and N‐acetyl cysteine
   (NAC) (Figure [78]S4A,B, Supporting Information), consistent with
   previous findings suggesting that SENP3 is a redox‐sensitive protein.^[
   [79]^11 , [80]^13 ^]

   Altogether, these results suggest that SENP3 is upregulated in human
   and murine AAA via an ROS‐dependent manner.

2.2. SENP3 Expression is Negatively Regulated by the E3 Ubiquitin Ligase
STUB1/CHIP

   Previous studies have reported that STUB1/CHIP, an essential E3
   ubiquitin ligase, facilitates the degradation of SENP3 under non‐stress
   conditions in tumor cells.^[ [81]^13 ^] Recently, STUB1/CHIP has been
   implicated in the pathogenesis of atherosclerosis via control of SIRT6
   stability in VSMCs.^[ [82]^14 ^] To explore the molecular mechanism by
   which SENP3 expression is regulated in AAA, we interrogated the
   expression profile of STUB1 in macrophages in AAA. We observed that the
   expression of STUB1 was notably decreased in BMDMs after AngII
   stimulation, in parallel with the upregulation of SENP3
   (Figure [83]S5A, Supporting Information). Immunofluorescence staining
   further confirms the downregulation of STUB1 in BMDMs after AngII
   stimulation (Figure [84]S5B, Supporting Information). The protein
   expression levels of STUB1 were further assessed in human and murine
   AAA specimens. Western blotting analysis suggested the dramatical
   downregulation of STUB1 in human AAA tissues (Figure [85]S5C,
   Supporting Information) and aneurysmal aortas from AngII‐infused
   ApoE^−/− mice (Figure [86]S5D, Supporting Information).

   We next investigated the effects of STUB1 knockdown and overexpression
   on SENP3 expression. We noted that small interfering RNA
   (siRNA)‐mediated STUB1 knockdown led to a robust increase of SENP3 in
   BMDMs (Figure [87]S5E, Supporting Information), whereas STUB1
   overexpression resulted in a strong reduction of SENP3 protein levels
   (Figure [88]S5F, Supporting Information). Moreover, we found that
   STUB1‐mediated SENP3 elimination was abolished following treatment with
   the 26S proteasome inhibitor MG132 in BMDMs (Figure [89]S5G, Supporting
   Information).

   To further explore the role of STUB1 in regulation of SENP3 stability,
   a co‐immunoprecipitation assay was performed to validate their
   interaction. As expected, the interaction between STUB1 and SENP3 was
   demonstrated by immunoprecipitation assay in BMDMs (Figure [90]S5H,I,
   Supporting Information). In addition, we found that overexpression of
   STUB1 triggered polyubiquitination of SENP3 (Figure [91]S5J, Supporting
   Information). These collective data suggest that the E3 ubiquitin
   ligase STUB1 targets SENP3 for ubiquitination and degradation.

2.3. Myeloid‐Specific SENP3 Deficiency Blunts AAA Formation in Mice

   To explore the functional effects of SENP3 on the pathogenesis of AAA,
   ApoE^−/−;Senp3^flox/flox and ApoE^−/−;Senp3^△Mø mice were
   subcutaneously injected with AngII or saline via osmotic minipumps for
   28 days (Figure [92]S6, Supporting Information; Figure [93] 2A).
   ApoE^−/−;Senp3^△Mø mice displayed better survival than
   ApoE^−/−;Senp3^flox/flox mice after 28 days of AngII‐infusion (93.3% in
   ApoE^−/−;Senp3^△Mø mice compared with 76.7% in ApoE^−/−;Senp3^flox/flox
   mice; p < 0.05) (Figure [94]2B). To non‐invasively assess the aortic
   enlargement after AngII infusion in vivo, we performed micro‐ultrasound
   imaging and found that AngII‐infused ApoE^−/−;Senp3^△Mø mice showed
   significantly smaller maximal internal diameters compared with
   ApoE^−/−;Senp3^flox/flox mice (Figure [95]2C,D). Upon dissection, we
   further confirmed the decreased maximal abdominal aortic diameter and
   ratio of aortic weight to body weight in ApoE^−/−;Senp3^△Mø mice,
   compared to ApoE^−/−;Senp3^flox/flox mice (Figure [96]2E–G). Notably,
   myeloid‐specific SENP3 deficiency was associated with a significantly
   decreased incidence of AAA formation after AngII infusion (40% [12 of
   30] of ApoE^−/−;Senp3^△Mø mice compared with 70% [21 of 30] of
   ApoE^−/−;Senp3^flox/flox mice; p < 0.05) (Figure [97]2H). Moreover, the
   incidence of aortic rupture in ApoE^−/−;Senp3^△Mø mice was also
   significantly decreased after AngII infusion compared with
   ApoE^−/−;Senp3^flox/flox mice (6.67% [2 of 30] of ApoE^−/−;Senp3^△Mø
   mice compared with 23.33% [7 of 30] of ApoE^−/−;Senp3^flox/flox mice; p
   < 0.05) (Figure [98]2I). Notably, the morphological analysis revealed
   that myeloid‐specific SENP3 deficiency reduced collagen deposition and
   elastic fiber breakage in abdominal aortic tissues after AngII‐infusion
   (Figure [99]2J–L).

Figure 2.

   Figure 2
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   Myeloid‐specific SENP3 deficiency represses AngII‐induced AAA
   formation. A) Schematic protocol: ApoE^−/−;Senp3^flox/flox and
   ApoE^−/−;Senp3^△Mø mice were subcutaneously injected with saline or
   AngII via a mini osmotic pump for 28 days (n = 30 per group). B)
   Survival curves in the indicated groups (n = 30 per group). Survival
   data were analyzed by the Kaplan–Meier method and compared using the
   log‐rank test. C,D) Representative images and quantification of the
   maximal abdominal aortic diameter monitored by micro‐ultrasound imaging
   (MUI) in the indicated groups (n = 20–30 per group). Scale bar: 5 mm.
   E) Representative images of macroscopic features of AAA formation in
   the indicated groups. Scale bar: 5 mm. F,G) Quantification of the
   maximal abdominal aortic diameter measured by a digital vernier caliper
   and ratio of aortic weight to body weight (BW) in the indicated groups
   (n = 20–30 per group). H,I) The incidences of AngII‐induced AAA (H) and
   aortic rupture (I) in the indicated groups (n = 30 per group). J)
   Representative images of suprarenal aortic sections stained with
   hematoxylin and eosin (H&E), Masson Trichrome, and Van Gieson after
   saline or Ang II infusion (n = 6 per group). Scale bar: 100 µm. K,L)
   Quantification of relative collagen content (K) and numbers of elastin
   breaks per vessel (L) in each group. Data represent mean ± SEM. p
   values were determined using two‐way ANOVA followed by Bonferroni
   post‐hoc test (D,F,G,K,L) and Fisher exact test (H,I). For all panels,
   *p < 0.05; **p < 0.01; ***p < 0.001.

   Female ApoE^−/−;Senp3^△Mø mice also exhibited a lower AAA incidence
   (Figure [101]S7A,B, Supporting Information) and smaller maximal
   abdominal aortic diameter (Figure [102]S7C, Supporting Information)
   after AngII infusion, compared to female ApoE^−/−;Senp3^flox/flox mice.
   However, no statistically significant differences were found in the
   incidence of ruptured aneurysms and ratio of aortic weight to body
   weight between ApoE^−/−;Senp3^flox/flox and ApoE^−/−;Senp3^△Mø female
   mice (Figure [103]S7D,E, Supporting Information). The above results
   reveal that myeloid‐specific SENP3 deficiency significantly ameliorates
   AngII‐induced AAA formation.

   To further investigate the role of SENP3 in the pathogenesis of AAA,
   Senp3^flox/flox and Senp3^△Mø mice were subjected to another
   experimental AAA formation model, involving the periaortic application
   of CaCl[2].^[ [104]^15 ^] Myeloid‐specific SENP3 deficiency
   significantly mitigated CaCl[2]‐induced AAA formation and reduced the
   maximal abdominal aortic diameter in both male and female mice
   (Figures [105]S8 and [106]S9, Supporting Information). Altogether,
   these results suggest that myeloid‐specific SENP3 deficiency inhibits
   AAA formation in both AngII‐ and CaCl[2]‐induced models.

2.4. SENP3 Deficiency Represses AAA Lesion Macrophage Infiltration and
Inflammatory Response

   In order to further interrogate the role of SENP3 in driving
   inflammatory vascular diseases, we performed high‐throughput RNA
   sequencing (RNA‐seq) of aortic tissues from ApoE^−/−;Senp3^flox/flox
   and ApoE^−/−;Senp3^△Mø mice infused with AngII for 28 days. We
   identified significant differences in the expression of genes
   associated with immunity and inflammation between the two groups
   (Figure [107] 3A–C). We therefore evaluated whether myeloid‐specific
   SENP3 deficiency ameliorates vascular inflammation in AAA.
   Immunofluorescence staining for CD68 revealed a significant reduction
   in macrophage infiltration in AAA tissues from ApoE^−/−;Senp3^△Mø mice
   compared to those from ApoE^−/−;Senp3^flox/flox mice (Figure [108]3D).
   To gain a deeper understanding of the inflammatory response, we
   assessed the expression of inflammatory factors using ELISA and
   immunohistochemistry. Our findings indicated that myeloid‐specific
   SENP3 deficiency significantly reduced the expression of inflammatory
   mediators (including IL‐6, TNF‐α, and MCP‐1) both in serum and
   abdominal aortic tissues after AngII infusion (Figure [109]3E,F).
   Furthermore, we found that SENP3‐deficient BMDMs exhibited decreased
   MCP‐1‐induced cell migration compared to control BMDMs
   (Figure [110]3G).

Figure 3.

   Figure 3
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   Myeloid‐specific SENP3 deficiency represses AAA Lesion macrophage
   infiltration and inflammatory response. A) The heatmap shows
   differentially expressed genes (DEGs) identified by RNA‐seq in aortic
   tissues from ApoE^−/−;Senp3^flox/flox and ApoE^−/−;Senp3^△Mø mice
   infused with AngII for 28 days. Each column represents an individual
   replicate, and each row represents an individual gene. Upregulated
   genes are shown in red, and downregulated genes are displayed in blue.
   N = 4 for each group. Genes with an adjusted p‐value < 0.01 and Fold
   Change ≥ 2 found by DESeq2 were assigned as differentially expressed.
   B) Volcano plot reveals the magnitude and significance of DEGs in
   aortic tissues from AngII‐infused ApoE^−/−;Senp3^flox/flox and
   ApoE^−/−;Senp3^△Mø mice. C) Gene Ontology (GO) pathway enrichment
   analysis of DEGs in aortic tissues from AngII‐infused
   ApoE^−/−;Senp3^flox/flox and ApoE^−/−;Senp3^△Mø mice. D) Representative
   images of immunofluorescence staining for CD68 (marker for macrophage)
   in abdominal aortic samples from ApoE^−/−;Senp3^flox/flox and
   ApoE^−/−;Senp3^△Mø mice infused with AngII for 28 days. Scale bar: 100
   µm. Quantification of fluorescence intensity of CD68 (n  =  4 for each
   group). E) Serum levels of IL‐6, TNF‐α, and MCP‐1 in
   ApoE^−/−;Senp3^flox/flox and ApoE^−/−;Senp3^△Mø mice infused with AngII
   for 28 days were assessed by ELISA. F) Immunohistochemistry analysis of
   IL‐6, TNF‐α, and MCP‐1 in abdominal aortas from
   ApoE^−/−;Senp3^flox/flox and ApoE^−/−;Senp3^△Mø mice infused with AngII
   for 28 days. Scale bar: 100 µm. G) Representative images of transwell
   migration assay and quantification of migrated cells (number/field) in
   BMDMs isolated from Senp3^flox/flox and Senp3^△Mø mice. Scale bar: 50
   µm. H) Representative images of dual immunofluorescence staining of
   MMP‐9 (green) and CD68 (red) in abdominal aortic samples from
   ApoE^−/−;Senp3^flox/flox and ApoE^−/−;Senp3^△Mø mice infused with AngII
   for 28 days. Where indicated, nuclei were counterstained with DAPI
   (blue). Scale bar: 100 µm. I) MMP9 protein levels were determined by
   western blot in abdominal aortic samples of ApoE^−/−;Senp3^flox/flox
   and ApoE^−/−;Senp3^△Mø mice infused with AngII or saline for 28 days.
   J) In situ zymography for gelatinase activity in abdominal aortic
   samples from ApoE^−/−;Senp3^flox/flox and ApoE^−/−;Senp3^△Mø mice
   infused with AngII for 28 days. Scale bar: 100 µm. Data represent mean
   ± SEM. p values were determined using the student's t‐test (D‐F,H‐J)
   and two‐way ANOVA followed by the Bonferroni post hoc test (G). For all
   panels, *p < 0.05; **p < 0.01; ***p < 0.001.

   Matrix metalloproteinases (MMPs) play a crucial role in the
   pathological process of AAA, especially VSMC‐derived MMP‐2 and
   macrophage‐derived MMP‐9.^[ [112]^16 ^] We therefore, examined the
   expression and activity of MMP‐9 in aortic tissues from
   ApoE^−/−;Senp3^flox/flox and ApoE^−/−;Senp3^△Mø mice infused with AngII
   or saline. Immunofluorescence staining and western blot analysis showed
   that 28 days of AngII‐infusion significantly upregulated the
   expressions of MMP‐9 in aortic tissues from ApoE^−/−;Senp3^flox/flox
   mice (Figure [113]3H,I). Additionally, MMP activity was assessed in
   vivo by an in situ zymography assay. As shown in Figure [114]3J,
   myeloid‐specific SENP3 deficiency decreased MMP activity in AAA.
   Altogether, these results suggest that SENP3 deficiency inhibits
   macrophage infiltration and inflammatory response during AAA formation.

2.5. SENP3 Deficiency Mitigates Ferroptosis in Macrophage

   Numerous studies have shown that macrophage ferroptosis is strongly
   implicated in aneurysmal and other inflammatory diseases.^[ [115]^8 ^.
   [116]^17 , [117]^18 ^] To gain additional insight into the potential
   mechanisms underlying how SENP3 regulates macrophage inflammation and
   AAA development, we speculate the potential role of SENP3 in macrophage
   ferroptosis in the pathogenesis of AAA. Our findings demonstrated the
   activation of ferroptosis in human aneurysms, as indicated by
   decreased protein levels of ferroptosis‐related proteins including
   GPX4, SLC7A11, and FTH1, and increased ACSL4 proteins (Figure [118]
   4A). In addition, we found excess iron deposition within the AAA
   lesions of human samples (Figure [119]4B), consistent with previous
   findings.^[ [120]^7 , [121]^19 ^] We therefore conducted a preliminary
   investigation into the impact of SENP3 on ferroptosis. The impact of
   myeloid‐specific SENP3 deficiency on ferroptosis in AngII‐induced AAA
   mouse model was investigated. Using RT‐qPCR on AAA specimens from
   ApoE^−/−;Senp3^flox/flox and ApoE^−/−;Senp3^△Mø mice, we found
   significant changes in ferroptosis indicators (Figure [122]4C). Western
   blot analysis showed no significant differences in protein levels
   of ferroptosis‐related proteins in aortic tissues between
   ApoE^−/−;Senp3^flox/flox and ApoE^−/−;Senp3^△Mø mice at baseline.
   However, the aortic tissues from ApoE^−/−;Senp3^△Mø mice exhibited
   partial restoration of aberrant protein levels of GPX4, ACSL4, SLC7A11,
   and FTH1 after 28 days of AngII infusion, compared with those in
   ApoE^−/−;Senp3^flox/flox mice (Figure [123]4D). Moreover, there was
   less iron deposition in aortic tissues from ApoE^−/−;Senp3^△Mø mice
   than in aortic tissues from ApoE^−/−;Senp3^flox/flox mice treated with
   AngII (Figure [124]4E), accompanied by significantly decreased ROS
   levels measured by DHE intensity (Figure [125]4F). In addition, the
   ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG)
   (GSH/GSSG) and levels of superoxide dismutase (SOD) and malondialdehyde
   (MDA) in aortic tissues from ApoE^−/−;Senp3^flox/flox and
   ApoE^−/−;Senp3^△Mø mice infused with AngII or saline were further
   measured. The results showed that GSH/GSSG and SOD levels were both
   decreased, whereas MDA levels were increased in aortic tissues of
   ApoE^−/−;Senp3^flox/flox mice after AngII infusion (Figure [126]4G).
   Notably, myeloid‐specific SENP3 deficiency significantly inhibited
   AngII‐induced decreases in GSH/GSSG and SOD levels, as well as
   increases in MDA production in aortic tissues (Figure [127]4G). The
   above results suggest that SENP3 deficiency mitigates ferroptosis in
   AAA.

Figure 4.

   Figure 4
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   SENP3 deficiency mitigates ferroptosis in macrophage. A) The protein
   levels of GPX4, ACSL4, SLC7A11, and FTH1 were determined by western
   blot in human AAA samples and adjacent nonaneurysmal control samples
   (non‐AAA) (n = 6 per group). B) Representative images and quantitative
   analysis of Berlin blue staining of iron in human non‐AAA and AAA
   samples (scale bar, 100 µm, n = 4). C,D) The mRNA levels and protein
   levels of GPX4, ACSL4, SLC7A11, and FTH1 were determined by RT‐qPCR (C)
   and western blot (D) in abdominal aortic samples from
   ApoE^−/−;Senp3^flox/flox and ApoE^−/−;Senp3^△Mø mice infused with AngII
   or saline for 28 days. E) Representative images and quantitative
   analysis of berlin blue staining of iron in abdominal aortic samples
   from ApoE^−/−;Senp3^flox/flox and ApoE^−/−;Senp3^△Mø mice infused with
   AngII or saline for 28 days (scale bar, 100 µm, n = 4). F)
   Representative images and quantitative analysis of dihydroethidium
   (DHE) staining in abdominal aortic samples from
   ApoE^−/−;Senp3^flox/flox and ApoE^−/−;Senp3^△Mø mice infused with AngII
   for 28 days (scale bar, 100 µm, n = 4). G) The ratio of
   reduced glutathione (GSH) to oxidized glutathione (GSSG) (GSH/GSSG) and
   levels of superoxide dismutase (SOD) and malondialdehyde (MDA) in
   aortic tissues from ApoE^−/−;Senp3^flox/flox and ApoE^−/−;Senp3^△Mø
   mice infused with AngII or saline for 28 days were measured. H) THP‐1
   were transfected with control siRNA (si‐con) and Senp3 siRNA (si‐Senp3)
   for 48 h. The protein levels of GPX4, ACSL4, SLC7A11, and FTH1 were
   determined by western blot. I,J) The levels of cellular ferrous iron
   (Fe^2+) and mitochondrial Fe^2+ were assessed by FerroOrange probes and
   Mito‐FerroGreen probes in BMDMs which were isolated from
   Senp3^flox/flox and Senp3^△Mø mice and stimulated with AngII (1
   µmol L^−1) or PBS for 24 h (scale bar, 100 µm, n = 4). K)
   Representative images of DHE staining in BMDMs isolated from
   Senp3^flox/flox and Senp3^△Mø mice and stimulated with AngII (1
   µmol L^−1) for 24 h (scale bar, 100 µm, n = 4). L) Representative
   images of Mito tracker (above) and electron micrograph of mitochondria
   (below) in BMDMs isolated from Senp3^flox/flox and Senp3^△Mø mice and
   stimulated with AngII (1 µmol L^−1) or PBS for 24 h (scale bar, 20 or
   0.5 µm, n = 4). Data represent mean ± SEM. p values were determined
   using the student's t‐test (A, B, F) and two‐way ANOVA followed by
   Bonferroni post‐hoc test (C–E,G–J). For all panels, *p < 0.05; **p <
   0.01; ***p < 0.001; ns: not significant (p > 0.05).

   To further verify the role of SENP3 in AngII‐induced ferroptosis,
   primary BMDMs were isolated from Senp3^flox/flox and Senp3^△Mø mice and
   were stimulated with AngII (1 µmol L^−1) for 24 h. Consistent with in
   vivo data, we found that SENP3‐deficient BMDMs exhibited increased GPX4
   and SLC7A11 proteins after AngII stimulation (Figure [129]S10A–D,
   Supporting Information), which was further demonstrated in human THP‐1
   cells (Figure [130]4H). In contrast, SENP3 overexpression exacerbated
   the changes of ferroptosis markers such as GPX4, ACSL4, SLC7A11, and
   FTH1 in BMDMs after AngII stimulation (Figure [131]S11, Supporting
   Information). In addition, the levels of cellular Fe^2+ and
   mitochondrial Fe^2+ were further assessed by FerroOrange and
   Mito‐FerroGreen probes in WT and SENP3‐deficient BMDMs. Our findings
   indicated that following AngII stimulation, the elevation of Fe^2+
   levels was significantly less pronounced in BMDMs isolated from
   Senp3^△Mø mice, compared to those from Senp3^flox/flox mice
   (Figure [132]4I,J). Moreover, SENP3 deficiency reversed the adverse
   events induced by AngII treatment, including increased cell death,
   ferrous iron (Fe^2+) level, ROS generation, and MDA production, as well
   as decreased SOD and GSH/GSSG levels in BMDMs in vitro
   (Figure [133]S10E–J, Supporting Information; Figure [134]4K),
   suggesting the protective effect of SENP3 deficiency on AngII‐induced
   ferroptosis in macrophages.

   It has been well demonstrated that the progression of ferroptosis
   predominantly induces mitochondrial morphological changes, including
   decrease in mitochondrial volume, increased mitochondrial membrane
   density, decreased or disappeared mitochondrial cristae, and rupture of
   the mitochondrial outer membrane.^[ [135]^20 , [136]^21 ^] We therefore
   investigated the impact of myeloid‐specific SENP3 deficiency on
   mitochondrial morphology. Utilizing confocal microscopy and
   transmission electron microscopy, we found that SENP3 deficiency
   partially restored mitochondrial ultrastructure damage in BMDMs after
   AngII stimulation (Figure [137]4L).

   Taken together, these data suggest that SENP3 deficiency mitigates
   ferroptosis in macrophage in vitro and in AAA in vivo.

2.6. SENP3 Regulates AAA by Targeting CTH to Regulate Ferroptosis Signals

   To clarify the mechanism by which SENP3 regulates AAA development, we
   reviewed the RNA‐seq data for aortic tissues from AngII‐infused
   ApoE^−/−;Senp3^flox/flox and ApoE^−/−;Senp3^△Mø mice. Bioinformatics
   analyses of differentially expressed genes suggested that PI3K/Akt
   signaling pathway was one of the most remarkable pathways enriched
   (data not shown). By utilizing IP‐MS quantitative data in conjunction
   with the STRING database based on the PI3K/Akt signaling pathways, as
   well as the macrophage gene set, 2 overlapping targets were identified,
   with CTH standing out as a crucial enzyme in the production of vascular
   H[2]S and a pivotal regulator of vascular function (Figure [138] 5A).
   Additionally, the established link between CTH and diverse biological
   processes such as macrophage inflammation, ferroptosis, and aneurysm
   development underscores its significance.^[ [139]^22 , [140]^23 ,
   [141]^24 , [142]^25 ^] Therefore, we examined whether SENP3 acts
   through CTH to modulate the AAA pathology. Our study revealed a
   reduction in the protein expression of CTH in human aneurysm tissues
   relative to non‐aneurysmal tissues (Figure [143]5B), aligning with
   existing literature.^[ [144]^26 ^] Consistently, CTH protein level was
   significantly decreased in aortic tissues from ApoE^−/−;Senp3^flox/flox
   after AngII infusion (Figure [145]5C). However, our results indicated
   that the downregulation of CTH in aortic tissues was partially reversed
   in ApoE^−/−;Senp3^△Mø mice after AngII infusion (Figure [146]5C).
   Additionally, our findings demonstrated that following AngII
   stimulation, the reduction in CTH expression was significantly restored
   in BMDMs isolated from Senp3^△Mø mice compared to those isolated from
   Senp3^flox/flox mice (Figure [147]5D).

Figure 5.

   Figure 5
   [148]Open in a new tab

   SENP3 regulates AAA by targeting CTH to regulate ferroptosis signals.
   A) By utilizing IP‐MS in conjunction with the STRING database based on
   PI3K/AKT signaling pathways, as well as macrophage gene set, two
   overlapping targets, CTH and Cox4i1, were identified. In the IP‐MS
   analysis results, the enrichment score of CTH was higher than that of
   Cox4i1. B) CTH protein levels were determined by western blot in human
   AAA samples and adjacent nonaneurysmal control samples (non‐AAA) (n = 6
   per group). C) CTH protein levels were determined by western blot in
   abdominal aortic samples of ApoE^−/−;Senp3^flox/flox and
   ApoE^−/−;Senp3^△Mø mice infused with AngII or saline for 28 days. D)
   CTH protein levels were determined by western blot in BMDMs which were
   isolated from Senp3^flox/flox and Senp3^△Mø mice and stimulated with
   AngII (1 µmol/L) for 180 min. E,F) Representative images of dual
   immunofluorescence staining for CTH (green) and CD68 (red) in abdominal
   aortic samples of ApoE^−/−;Senp3^flox/flox and ApoE^−/−;Senp3^△Mø mice
   infused with AngII for 28 days. Where indicated, nuclei were
   counterstained with DAPI (blue). G,H) Representative images of dual
   immunofluorescence staining for CTH (green) and CD68 (red) in BMDMs
   which were isolated from Senp3^flox/flox and Senp3^△Mø mice and
   stimulated with AngII (1 µmol L^−1) for 180 min. Where indicated,
   nuclei were counterstained with DAPI (blue). I) The plasma levels of
   H[2]S in ApoE^−/−;Senp3^flox/flox and ApoE^−/−;Senp3^△Mø mice infused
   with saline or AngII for 28 days were determined (n = 6 for each
   group). J) H[2]S concentrations were determined for the cellular
   homogenate of BMDMs which were isolated from Senp3^flox/flox and
   Senp3^△Mø mice and stimulated with AngII (1 µmol L^−1) for 24 h. K)
   BMDMs were transfected with control siRNA (si‐NC) and CTH siRNA
   (si‐CTH) for 48 h. The knockout efficiency of CTH was verified by
   western blot. L) The protein levels of GPX4, ACSL4, SLC7A11, and FTH1
   were determined by western blot in BMDMs which were isolated from
   Senp3^flox/flox and Senp3^△Mø mice and were further transfected with
   si‐NC and si‐CTH for 48 h before stimulation with AngII (1 µmol L^−1)
   for 24 h. M) BMDMs isolated from Senp3^flox/flox and Senp3^△Mø mice
   were transfected with si‐NC and si‐CTH for 48 h before stimulation with
   AngII (1 µmol L^−1) for 24 h. The mRNA levels of IL‐1β, TNF‐α, CCL2,
   and IL‐6 were determined by RT‐qPCR. p values were determined using
   student's t‐test (B, F, H, and K) and two‐way ANOVA followed by
   Bonferroni post‐hoc test (C,D,I,J,L,M). For all panels, *p < 0.05; **p
   < 0.01; ***p < 0.001; ns: not significant (p > 0.05).

   As CTH functions as a pivotal enzyme in the biosynthesis of vascular
   H[2]S,^[ [149]^27 ^] we assessed H[2]S concentrations across various
   experimental groups in both in vivo and in vitro systems. We found that
   H[2]S levels were dramatically decreased in plasma of
   ApoE^−/−;Senp3^flox/flox mice after AngII infusion (Figure [150]5I).
   Myeloid‐specific SENP3 deficiency significantly reversed the decreases
   in H[2]S levels after AngII infusion (Figure [151]5I). This was further
   confirmed in primary BMDMs in vitro (Figure [152]5J). Collectively, the
   above results suggest that SENP3 negatively regulates CTH expression
   and H[2]S production.

   To investigate the potential involvement of CTH in mediating the
   effects of SENP3 deficiency on ferroptosis, we knocked down CTH
   expression by transfecting BMDMs with siRNA targeting CTH
   (Figure [153]5K). We observed that CTH knockdown significantly
   abolished SENP3 deficiency‐induced increases in ferroptosis‐related
   proteins GPX4, SLC7A11, and FTH1 and decreases in ACSL4 proteins in
   BMDMs after AngII stimulation (Figure [154]5L). Consistently, we found
   that CTH knockdown blocked the effect of SENP3 deficiency on
   AngII‐induced inflammatory cytokine production in BMDMs
   (Figure [155]5M). Moreover, we further demonstrated that
   pharmacological inhibition of CTH with DL‐Propargylglycine (PAG) also
   attenuated the protective effect of SENP3 deficiency on AngII‐induced
   ferroptosis. Specifically, SENP3 deficiency decreased cell death, Fe^2+
   level, ROS generation, and MDA production, as well as increased SOD and
   GSH/GSSG levels in BMDMs after AngII stimulation. Nevertheless, the
   protective effects were attenuated in the presence of PAG
   (Figure [156]S12, Supporting Information). These findings suggest that
   SENP3 deficiency may modulate ferroptosis through its regulation of CTH
   expression.

2.7. SENP3 De‐SUMOylates CTH and Regulates its Protein Stability

   Given that SENP3 is a SUMO2/3‐specific protease, it is hypothesized
   that CTH may undergo SUMOylation, while SENP3 may catalyze the
   deSUMOylation of CTH. As shown in Figure [157] 6A, CTH SUMOylation was
   detected by immunoprecipitation in HEK‐293T cells co‐expressed with
   Flag‐CTH and HA‐SUMO3, while SENP3 deconjugated SUMO‐3 from CTH. The
   interaction between CTH and SENP3 was further confirmed by
   co‐immunoprecipitation assay in exogenous setting and endogenous system
   of BMDMs (Figure [158]6B–D). These findings imply that SENP3
   de‐SUMOylates CTH via a direct interaction.

Figure 6.

   Figure 6
   [159]Open in a new tab

   SENP3 de‐SUMOylates CTH and regulates its protein stability. A) 293T
   cells were transfected with Flag‐CTH, HA‐SUMO3, GFP‐SENP3, and Ubc‐9
   for 48 h. The SUMOylation of Flag‐CTH was determined by the
   immunoprecipitation (IP) assay using Flag beads and western blot using
   anti‐Flag, anti‐HA, and anti‐GAPDH antibodies. B) Exogenous interaction
   between SENP3 and CTH was determined by co‐immunoprecipitation (co‐IP)
   using GFP‐beads. C,D) The endogenous interaction between SENP3 and CTH
   was determined in BMDMs stimulated with AngII by coimmunoprecipitation
   assay using antibody against SENP3 or CTH. E,F) SUMOplotTM Analysis
   Program online tool ([160]https://www.abcepta.com/sumoplot, accessed on
   11 February 2023), JASSA, and GPS‐SUMO were employed to predict the
   SUMOylation sites of CTH. G) Structure of CTH protein and site of K361.
   H) 293T cells were transfected with Flag‐CTH/WT or Flag‐CTH/K361R,
   HA‐SUMO3, and Ubc‐9 for 48 h. The SUMOylation of Flag‐CTH was
   determined by the IP assay using Flag beads and western blot using
   anti‐Flag, anti‐HA, and anti‐GAPDH antibodies. I) 293T cells were
   transfected with Flag‐CTH and increasing amounts of GFP‐SENP3 for 48 h.
   The protein levels of Flag‐CTH, GFP‐SENP3, and GAPDH in whole cell
   lysates were determined by western blot with anti‐Flag, anti‐GFP, and
   anti‐GAPDH antibodies. J) BMDMs were infected with increasing amounts
   of adenovirus expressing HA‐SENP3 for 48 h. The levels of CTH and
   HA‐SENP3 in whole cell lysates were determined by western blot with
   anti‐CTH, anti‐HA, anti‐SENP3, and anti‐GAPDH antibodies. K) 293T cells
   were transfected with control siRNA (si‐NC) and SENP3 siRNA (si‐SENP3)
   for 48 h. Lysates were prepared and analyzed by western blot. L) 293T
   cells were transfected with Flag‐CTH and equal amounts of PCDNA or
   GFP‐SENP3 or GFP‐SENP3/C532A for 48 h. Lysates were prepared and
   analyzed by western blot. M) 293T cells were transfected with Flag‐CTH
   and increasing amounts of GFP‐SENP3 for 48 h, in the presence or
   absence of MG132 (10 µmol L^−1) for the last 10 h. Lysates were
   prepared and analyzed by western blot. N) 293T cells were transfected
   with Flag‐CTH and PCDNA or GFP‐SENP3 for 36 h and were subsequently
   exposed to the protein synthesis inhibitor cycloheximide (CHX) for the
   indicated time. Lysates were prepared and analyzed by western blot. The
   relative level of CTH was evaluated by densitometry and normalized to
   GAPDH. O) 293T cells were transfected with Flag‐CTH/WT or
   Flag‐CTH/K361R and increasing amounts of GFP‐SENP3 for 48 h. The levels
   of Flag‐CTH in whole‐cell lysates were determined by western blot with
   anti‐Flag, anti‐GFP, and anti‐GAPDH antibodies.

   To investigate the amino acid residues targeted for SUMOylation on CTH,
   we employed various computational‐system‐based software including
   SUMOplotTM, JASSA, and GPS‐SUMO to predict potential SUMOylation sites.
   As shown in Figure [161]6E–G, the analysis of SUMOylation sites
   predicted by the three software sets revealed that only the K361 site
   exhibited overlap. We therefore mutated the predicted SUMOylation site
   K361 to arginine (R), constructing CTH site‐specific single‐point
   mutant CTH‐K361R. The Flag beads pull‐down assay revealed that the
   SUMOylation of CTH was partially abolished by the K361R single‐point
   mutation, indicating that K361 is the main SUMOylation site in CTH
   (Figure [162]6H). Collectively, our results suggest that CTH is
   SUMOylated at K361.

   It has been well demonstrated that SUMOylation can regulate protein
   stability of substrate proteins.^[ [163]^11 , [164]^12 , [165]^28 ^] We
   then investigated whether SENP3 and SUMOylation regulate CTH protein
   stability. We found that the protein level of CTH was dose‐dependently
   decreased by SENP3 overexpression (Figure [166]6I,J). Knockdown of
   SENP3 in 293T cells led to upregulation of CTH expression
   (Figure [167]6K). As cysteine 532 of SENP3 dominates its enzymatic
   activity, we overexpressed SENP3‐C532A, an enzymally inactive mutant,
   and observed that the protein level of CTH was decreased by SENP3‐WT,
   but not SENP3‐C532A (Figure [168]6L). This result reveals that the
   enzymatic activity of SENP3 is essential for the modulation of CTH
   protein levels. Moreover, we found that SENP3‐mediated CTH elimination
   was abolished following treatment with the 26S proteasome inhibitor
   MG132 (Figure [169]6M). To further demonstrate this point of view,
   protein synthesis inhibitor cycloheximide (CHX) was used to suppress
   protein biosynthesis. The half‐life of exogenous CTH was markedly
   shortened in cells overexpressing SENP3 (Figure [170]6N). Consistent
   with these results, SENP3‐mediated CTH downregulation was abolished in
   the CTH‐K361R mutant form (Figure [171]6O). Collectively, these
   findings suggest that SENP3 facilitates the degradation of CTH through
   a proteasome‐dependent mechanism.

   As SENP3 acts as a SUMO2/3‐specific protease, we then examined the
   effect of SUMOylation (SUMO‐3) on CTH expression. We found that the
   protein level of CTH was dose‐dependently increased by SUMO‐3
   overexpression, which was abolished following treatment with the MG132
   (Figure [172]S13A,B, Supporting Information). In addition, the elevated
   protein level of CTH after SUMO‐3 overexpression was compromised by
   CTH/K361R mutant (Figure [173]S13C, Supporting Information). To further
   explore the role of SUMOylation in modulating CTH stability, cells were
   treated with the CHX. Co‐expression of SUMO‐3 with CTH increased the
   half‐life of CTH (Figure [174]S13D, Supporting Information). By
   co‐transfecting CTH, SUMO3, and SENP3, we further confirmed that SUMO‐3
   promoted CTH protein stability, whereas SENP3 facilitated its
   degradation (Figure [175]S13E, Supporting Information). Altogether,
   these results suggest that SENP3 de‐SUMOylates CTH and regulates its
   protein stability.

2.8. CTH Inhibitor Counteracts the Protective Effect of SENP3 Deficiency on
AAA

   To further explore the contribution of CTH signaling to the function of
   SENP3 in AAA pathology, saline‐ or AngII‐infused
   ApoE^−/−;Senp3^flox/flox and ApoE^−/−;Senp3^△Mø mice were treated with
   PAG, a selective inhibitor of CTH, at a dose of 50 mg kg^−1 day^−1 for
   28 days (Figure [176] 7A). As illustrated in Figure [177]7B, PAG
   treatment exacerbated the mortality of ApoE^−/−;Senp3^flox/flox and
   ApoE^−/−;Senp3^△Mø mice after AngII‐infusion (20% versus 35% for
   ApoE^−/−;Senp3^flox/flox mice; p < 0.05; 5% versus 25% for
   ApoE^−/−;Senp3^△Mø mice; p < 0.05). However, there was no significant
   difference in the survival rates between AngII‐infused
   ApoE^−/−;Senp3^flox/flox and ApoE^−/−;Senp3^△Mø mice that received PAG
   treatment (Figure [178]7B) (65% vs 75%; p > 0.05). Moreover, we found
   that PAG treatment exacerbated aortic enlargement (Figure [179]7C–F)
   and increased the incidences of AAA and aortic rupture
   (Figure [180]7G,H) in ApoE^−/−;Senp3^flox/flox and ApoE^−/−;Senp3^△Mø
   mice after AngII‐infusion. Notably, we found that the reduced aortic
   enlargement and reduced incidences of AAA and aortic rupture in
   ApoE^−/−;Senp3^△Mø mice after AngII‐infusion in comparison with those
   in ApoE^−/−;Senp3^flox/flox mice after AngII‐infusion, were effectively
   reversed by PAG treatment (Figure [181]7C–H). In Addition, the better
   aortic structural integrity and less collagen accumulation and elastic
   fiber breakage in ApoE^−/−;Senp3^△Mø mice after AngII‐infusion,
   compared with those in ApoE^−/−;Senp3^flox/flox mice after
   AngII‐infusion, were effectively reversed by PAG treatment
   (Figure [182]7I–K), suggesting that pharmacological CTH inhibition
   counteracts the protective effect of SENP3 deficiency on AAA. Taken
   together, these findings indicate that the protective effect of SENP3
   deficiency in AAA depends on CTH signaling.

Figure 7.

   Figure 7
   [183]Open in a new tab

   CTH inhibitor counteracts the protective effect of SENP3 deficiency on
   AAA. A) Schematic protocol: ApoE^−/−;Senp3^flox/flox and
   ApoE^−/−;Senp3^△Mø mice were infused with saline or AngII (1.44
   mg kg^−1 d^−1) and intraperitoneally injected with DL‐Propargylglycine
   (PAG) (50 mg kg^−1 d^−1) for 28 days (n = 20 per group). B) Survival
   curves in the indicated groups (n = 20 per group). Survival data were
   analyzed by the Kaplan–Meier method and compared using the log‐rank
   test. C,D) Representative images and quantification of the maximal
   abdominal aortic diameter monitored by micro‐ultrasound imaging in each
   group. Scale bar: 5 mm. E) Representative images of the abdominal aorta
   visualized by macroscopic examination in the indicated groups. Scale
   bar: 5 mm. F) Quantification of the maximal abdominal aortic diameter
   measured by a digital vernier caliper for each group. G,H) The
   incidences of AngII‐induced AAA (G) and aortic rupture (H) in the
   indicated groups. Data were analyzed by a Fisher exact test. I)
   Representative images of suprarenal aortic sections stained with
   hematoxylin and eosin (H&E), Masson Trichrome, and Van Gieson in each
   group. Scale bar: 100 µm. J,K) Quantification of relative collagen
   content (J) and numbers of elastin breaks per vessel (K) in each group.
   Data represents mean ± SEM. p values were determined using two‐way
   ANOVA followed by Bonferroni post‐hoc test (D,F,J,K) and Fisher exact
   test (G,H). For all panels, *p < 0.05; **p < 0.01; ***p < 0.001;
   ns: not significant (p > 0.05).

2.9. Supplementation with the H[2]S Donor ATB346 Protects Against AAA
Formation

   As a crucial inflammatory mediator in macrophages, H[2]S directly or
   indirectly modulates various pathophysiological process in
   cardiovascular disorders such as aneurysms, atherosclerosis, and
   pulmonary hypertension.^[ [184]^29 ^] The promise of H[2]S‐based
   therapeutics is now being demonstrated in clinical trials, with ATB‐346
   emerging as the most advanced compound under scrutiny.^[ [185]^30 ^]
   ATB‐346 is a hydrogen sulfide‐releasing non‐steroidal anti‐inflammatory
   drug derived from naproxen, which exhibits anti‐inflammatory effects in
   conditions such as gastrointestinal ulcers/hemorrhages, spinal cord
   injury, postoperative ileus, and periodontitis.^[ [186]^31 , [187]^32 ,
   [188]^33 , [189]^34 ^] Thus, we wondered whether the impaired H[2]S
   cycle in AAAs would be reactivated by supplementing the key H[2]S donor
   ATB346.

   To verify this hypothesis, saline‐ or AngII‐infused
   ApoE^−/−;Senp3^flox/flox and ApoE^−/−;Senp3^△Mø mice were
   intragastrically administered with ATB346 at a dose of 16 mg kg^−1 for
   28 days. ATB346 supplementation resulted in improved survival in
   AngII‐infused ApoE^−/−;Senp3^flox/flox mice (75% vs 95%; p < 0.05), but
   had no significant effects on survival rate in AngII‐infused
   ApoE^−/−;Senp3^△Mø mice (95% vs 95%; p > 0.05) (Figure [190] 8A,B).
   Furthermore, our findings indicated that ATB346 improved the maximal
   internal diameters in AngII‐infused ApoE^−/−;Senp3^flox/flox mice,
   while showing no significant effects in AngII‐infused
   ApoE^−/−;Senp3^△Mø mice (Figure [191]8C–F). Although ATB346
   supplementation did not significantly decreased AAA incidence in
   AngII‐infused ApoE^−/−;Senp3^flox/flox mice (Figure [192]7G), it
   significantly decreased the incidence of aortic rupture
   (Figure [193]7H) in AngII‐infused ApoE^−/−;Senp3^flox/flox mice,
   suggesting that ATB346 supplementation improved AAA pathology in
   ApoE^−/−;Senp3^flox/flox mice to some extent. Moreover, ATB346
   supplementation significantly reduced pathological collagen
   accumulation and elastic fiber breakage in abdominal aortic tissues in
   AngII‐infused ApoE^−/−;Senp3^flox/flox mice (Figure [194]8I–K). It is
   noteworthy that the protective effects of ATB346 on AAA were completely
   abolished in ApoE^−/−;Senp3^△Mø mice (Figure [195]8). In addition, in
   in vitro system of primary BMDMs, we demonstrated that ATB346 improved
   AngII‐induced ferroptosis (Figure [196]S14, Supporting Information).
   Altogether, these results suggest that supplementation with ATB3466
   protects against AAA injuries. Moreover, they further confirm our
   conclusion that SENP3 regulates AAA by targeting CTH to regulate
   ferroptosis signals.

Figure 8.

   Figure 8
   [197]Open in a new tab

   Supplementation with the H[2]S donor ATB346 protects against AAA
   formation. A) Schematic protocol: ApoE^−/−;Senp3^flox/flox and
   ApoE^−/−;Senp3^△Mø mice were infused with saline or AngII (1.44
   mg kg^−1 d^−1) and intragastrically injected with ATB346 (16 mg kg^−1)
   for 28 days (n = 20 per group). B) Survival curves in the indicated
   groups (n = 20 per group). Survival data were analyzed by the
   Kaplan‐Meier method and compared using the log‐rank test. C,D)
   Representative images and quantification of the maximal abdominal
   aortic diameter monitored by micro‐ultrasound imaging in each group.
   Scale bar: 5 mm. E) Representative images of the abdominal aorta
   visualized by macroscopic examination in the indicated groups. Scale
   bar: 5 mm. F) Quantification of the maximal abdominal aortic diameter
   by a digital vernier caliper for each group. Data were analyzed by
   two‐way ANOVA followed by the Bonferroni post hoc test. G,H) The
   incidences of AngII‐induced AAA (G) and aortic rupture (H) in the
   indicated groups. I) Representative images of suprarenal aortic
   sections stained with hematoxylin and eosin (H&E), Masson Trichrome,
   and Van Gieson in each group. Scale bar: 100 µm. J,K) Quantification of
   relative collagen content (J) and numbers of elastin breaks per vessel
   (K) in each group. Data represent mean ± SEM. p values were determined
   using two‐way ANOVA followed by Bonferroni post‐hoc test (D,F,J,K) and
   Fisher exact test (G.H). For all panels, *p < 0.05; **p < 0.01; ***p <
   0.001; ns: not significant (p > 0.05).

3. Discussion

   In this present study, we identified a novel role of the SUMO‐specific
   protease SENP3 in the pathogenesis of AAA. SENP3 protein expression was
   upregulated in macrophages in both human and murine AAA specimens.
   Myeloid‐specific SENP3 deficiency protected mice against AAA formation
   in both AngII‐ and CaCl[2]‐induced mouse models. Mechanistically, we
   demonstrated that CTH, a critical enzyme involved in H[2]S production
   within the vessels, as a novel substrate of SENP3, which mediated the
   regulatory effects of SENP3 on ferroptosis and inflammatory programs in
   macrophages. CTH was SUMOylated at Lysine 361 and could be
   de‐SUMOylated by SENP3. SENP3 facilitated the proteasome‐dependent
   degradation of CTH. In addition, we further provide evidence
   demonstrating that pharmacological inhibition of CTH counteracted the
   protective effect of SENP3 deficiency on AAA formation. Additionally,
   supplementation with the H[2]S donor ATB346 protected against AAA
   formation. Overall, the results of the present study provided the first
   evidence for a novel regulatory SENP3/CTH/ferroptosis signaling axis in
   AAA formation and progression.

   SUMOylation has emerged as an important PTM that regulates protein
   activity, subcellular localization, stability, and protein‐protein
   interactions.^[ [198]^35 ^] SUMOylation is a dynamic and
   reversible process, which is catalyzed by SUMO‐specific ligases
   and reversed by a family of SENPs.^[ [199]^10 ^] SENP3, one of the six
   SENP family members (SENP1–3 and 5–7), has been known as an important
   regulator of oxidative stress, inflammation, and programmed cell
   death.^[ [200]^36 ^] Recent studies have uncovered several new
   functions of SENP3 (i.e., regulating inflammatory responses, ribosome
   biogenesis, and mitochondrial damage) in the pathogenesis of cancer,
   ischemia‐reperfusion injury, autoimmune and inflammatory diseases.^[
   [201]^37 , [202]^38 , [203]^39 ^] Accumulating evidence has shown that
   SENP3 regulates cardiovascular physiology/pathology.^[ [204]^11 ,
   [205]^40 , [206]^41 ^] We have previously demonstrated that SENP3
   regulates VSMC function and low‐shear stress‐induced vascular
   remodeling.^[ [207]^11 ^] However, the role of SENP3 in the
   pathogenesis of AAA has never been investigated. In this present study,
   we found that SENP3 protein was upregulated in both human and murine
   AAA specimens in vivo and in BMDMs in vitro after AngII treatment via a
   ROS‐dependent manner. Myeloid‐specific deletion of SENP3 protected mice
   against AngII‐ and CaCl[2]‐induced AAA formation. These findings
   provide compelling evidence of a previously unrecognized role of SENP3
   as a critical regulator in the pathogenesis of AAA.

   Ferroptosis is an iron‐dependent form of nonapoptotic programmed cell
   death that is distinct from apoptosis, necrosis, and autophagy^[
   [208]^42 , [209]^43 ^] and has been strongly implicated in multiple
   diseases and pathological conditions, such as degenerative diseases,
   ischemic disorders, and cardiovascular disease.^[ [210]^42 ^] Numerous
   previous studies have suggested that inhibition of ferroptosis
   effectively ameliorated the occurrence and development of AAA.^[
   [211]^7 , [212]^8 ^] SENP3 has been previously demonstrated to regulate
   inflammatory, mitochondrial metabolism, and programmed cell death in
   several organs and cells.^[ [213]^11 , [214]^12 , [215]^39 ^] Here we
   provide first evidence demonstrating that SENP3 deficiency mitigated
   AngII‐induced ferroptosis in macrophage in vitro and in vivo. Moreover,
   we identified CTH, a critical enzyme involved in H[2]S production, as a
   novel target protein of SENP3 that mediated the regulatory effects of
   SENP3 on ferroptosis and inflammatory programs in macrophages in AAA.
   CTH and the H[2]S signaling pathway have been well‐identified as key
   regulators of vascular function. For example, CTH deletion exacerbates
   the progression of AAA and decreased plasma H[2]S levels are associated
   with an increased risk of AAA.^[ [216]^26 ^] Deficiency of CTH promotes
   aortic elastolysis and medial degeneration in aged mice.^[ [217]^44 ^]
   Inhibition of CTH dampens vasoconstriction in mouse and human
   intracerebral arterioles.^[ [218]^45 ^] Our research indicated that the
   protective effects of SENP3 deficiency on ferroptosis and AAA
   pathogenesis were mediated by CTH signaling. We found that CTH was
   SUMOylated at lysine 361, which could be deconjugated by SENP3. SUMO‐3
   promoted CTH protein stability, whereas SENP3 facilitated its
   proteasome‐dependent degradation. This study identifies the SENP3/CTH
   axis as a potential novel target for regulating inflammation and
   ferroptosis in AAA treatment.

   In recent years, the role of H[2]S in cardiovascular systems has
   received increasing attention due to its versatile functions. As a
   crucial inflammatory mediator in macrophages, H[2]S directly or
   indirectly modulates various cellular activities, such as phagocytosis,
   migration, cytokine production, redox homeostasis, and energy
   metabolism, suggesting it as a promising therapeutic target for
   inflammatory disease. ATB‐346 is a H[2]S‐donating derivative of the
   non‐steroidal anti‐inflammatory compound naproxen. Previous preclinical
   studies have demonstrated that ATB‐346 exhibits anti‐inflammatory
   effects in conditions such as gastrointestinal ulcers/haemorrhages,
   spinal cord injury, postoperative ileus, and periodontitis.^[ [219]^31
   , [220]^32 , [221]^33 , [222]^34 ^] Moreover, the robust
   anti‐inflammatory properties of ATB‐346 have been validated in human
   studies of inflammation.^[ [223]^46 , [224]^47 ^] Nevertheless, there
   are currently no evidence about ATB‐346 being applied in AAA. Based on
   our experimental findings and given the importance of H[2]S in the
   physiological and pathological regulation of vasculature, we
   tentatively applied ATB346 in mouse AAA model. We were therefore
   surprised to find that supplementation with ATB346 was beneficial to
   slow down the progression of AAA. However, further investigations to
   confirm the treatment values are essential for bringing these
   interventions to the clinic.

   In conclusion, we demonstrate that SENP3 accumulates in macrophages of
   aneurysmal aortic tissues, which promotes macrophage ferroptosis and
   inflammation via regulation of CTH De‐SUMOylation. The findings provide
   us with a new perspective for understanding the role of
   SENP3/CTH/ferroptosis axis in the pathogenesis of AAA. A better
   understanding of the role of SENP3 and CTH in AAA may ultimately lead
   to new approaches to prevent and treat aortic aneurysmal diseases.

4. Experimental Section

Human Tissue Specimens

   All human aneurysm specimens utilized in this research were obtained
   from patients who had undergone open abdominal aortic repair, while
   control samples were extracted from non‐dilated aortic tissue from the
   same individuals. Patient consent was obtained for the use of tissue
   samples. The collection and utilization of human specimens adhered to
   the protocol approved by the Institutional Review Board of Shanghai
   Jiao Tong University. Participants were recruited with informed consent
   approved by the Ethics Committee of Shanghai Chest Hospital, Shanghai
   Jiao Tong University School of Medicine (approval IS21014).

Animal Experiments

   Senp3^flox/flox mice were kindly provided by Prof. Jing Yi from
   Shanghai Jiao Tong University School of Medicine, Shanghai, China.
   Lyz2‐Cre mice (Cat. NO. NM‐KI‐215037), ApoE^−/−  mice (Cat. NO.
   NM‐KO‐190565), and C57BL/6J mice were purchased from Shanghai Model
   Organisms Center, Inc. Senp3^flox/flox mice were intercrossed with
   Lyz2‐Cre mice to generate Senp3^flox/flox ;Lyz2‐Cre (Senp3^△Mø ) mice,
   which were further bred with ApoE^−/− mice to establish
   ApoE^−/−;Senp3^flox/flox ;Lyz2‐Cre (ApoE^−/−;Senp3^△Mø ) mice. Male and
   female mice, aged 8–12 weeks, were utilized in the study. Mice were
   maintained in controlled conditions of 24 ± 2 °C and 40 ± 5% humidity,
   subjected to a 12‐h light/dark cycle, and allowed free access to water
   and food. The animal protocol employed in this research was sanctioned
   by the Ethics Committee of Shanghai Chest Hospital (Permit Number:
   KS24062) and adhered to the National Institutes of Health Guidelines
   for the Care and Use of Laboratory Animals.

Statistical Analysis

   All statistical analyses were performed using GraphPad Prism 6 or SPSS
   version 20.0. Continuous variables were expressed as mean ± standard
   error of the mean (SEM), and categorical variables were presented as
   numbers or percentages. The Shapiro‐Wilk test was utilized to evaluate
   the normality of the data distribution. For comparisons between two
   groups, the student's t‐test was applied for variables with equal
   variances, while the unequal variance t‐test was used for variables
   with unequal variances, provided the data were normally distributed.
   For variables that did not follow a normal distribution, the
   Mann–Whitney U test was employed. For comparisons involving more than
   two groups, the Brown‐Forsythe test was initially employed to assess
   the homogeneity of variances. If the data satisfied the assumption of
   equal variances, a one‐way ANOVA was conducted, followed by a post hoc
   analysis using the Bonferroni method to adjust for multiple
   comparisons. Otherwise, a Welch ANOVA test was performed, followed by a
   post hoc analysis using the Tamhane T2 method. Two‐way ANOVA followed
   by Bonferroni post hoc analysis was used when >2 groups and variables
   were compared. Bivariate comparisons of categorical variables were
   performed with the χ^2 test. Survival was estimated by the Kaplan‐Meier
   method, and differences were evaluated using a stratified log‐rank
   test. A value of p < 0.05 was considered statistically significant.

   More details can be found in Supporting Information.

Conflict of Interest

   The authors declare no conflict of interest.

Author Contributions

   L.C., Z.C., D.X., and Y.S. contributed equally to this work. L.C.,
   Z.C., D.X., and Y.S. designed and executed the experiments, and
   analyzed the data. L.C. wrote the manuscript. Q.X., M.L., Y.J., Y.H.,
   F.L., G.Z., X.W., H.H., and L.F. executed the experiments and provided
   technical assistance. F.Z. provided technical assistance and acquired
   the funding. Z.C., Q.S., and B.H. acquired the funding, conceived and
   designed the study, and revised the manuscript. All authors have read
   and agreed to the published version of the manuscript.

Supporting information

   Supporting Information
   [225]ADVS-12-2414500-s001.pdf^ (8MB, pdf)

Acknowledgements