Repeated Social Defeat Enhances CaCl2-Induced Abdominal Aortic Aneurysm Expansion by Inhibiting the Early Fibrotic Response via the MAPK-MKP-1 Pathway

Depression is an independent risk factor for cardiovascular disease and is significantly associated with the prevalence of abdominal aortic aneurysm (AAA). We investigated the effect of repeated social defeat (RSD) on AAA development. Eight-week-old male wild-type mice were exposed to RSD by being housed with larger CD-1 mice in a shared cage. They were subjected to vigorous physical contact. After the confirmation of depressive-like behavior, calcium chloride was applied to the infrarenal aorta of the mice. At one week, AAA development was comparable between the defeated and control mice, without any differences being observed in the accumulated macrophages or in the matrix metalloproteinase activity. At two weeks, the maximum diameter and circumference of the aneurysm were significantly increased in the defeated mice, and a significant decrease in periaortic fibrosis was also observed. Consistently, the phosphorylation of the extracellular signal-regulated kinase and the incorporation of 5-bromo-2′-deoxyuridine in the primarily cultured aortic vascular smooth muscle cells were significantly reduced in the defeated mice, which was accompanied by a substantial increase in mitogen-activated protein kinase phosphatase-1 (MKP-1). The MKP-1 mRNA and protein expression levels during AAA were much higher in the defeated mice than they were in the control mice. Our findings demonstrate that RSD enhances AAA development by suppressing periaortic fibrosis after an acute inflammatory response and imply novel mechanisms that are associated with depression-related AAA development.


Introduction
Mental disorders are an important cause of debility worldwide and are a main contributor to comprehensive disease burden and represent substantial causes of death [1]. Meta-analyses have revealed that depression is related to a greater risk of cardiovascular disease and subsequent outcomes after myocardial infarction [2][3][4][5]. There is increasing evidence that depression is causally connected to the development of atherosclerotic cardiovascular disease (CVD) through the integration of various factors; however, the detailed mechanisms of depression-related CVD development remain to be fully elucidated [6][7][8].
Industry Co., Ltd., Osaka, Japan). In the social interaction test, the time spent in the interaction zone when the target was absent or present was recorded using a CCD video camera. The social interaction ratio (SIR) was calculated by dividing the interaction time spent in the presence of the target by the time spent in the absence of the target. The RSD-exposed mice with an SIR less than 1.0 were certified as defeated mice with depression-like behavior, while non-exposed mice exhibiting an SIR that was not less than 1.0 were certified as control mice.

Mouse Aneurysm Model
After the behavioral analysis, AAAs were developed via the periaortic application of 0.5 m calcium chloride (CaCl 2 ), as previously described [22]. Ten-week-old mice were anesthetized by isoflurane (2%, 0.2 mL/min) using an anesthetic instrument (PITa-Quark; Sanko Manufacturing Co., Ltd., Saitama, Japan) during the surgery. The depth of the anesthesia was examined via the lack of a tail pinch response and was carefully monitored during the surgery. A laparotomy was performed under sterile conditions with the support of a stereomicroscope (Leica Microsystems K.K., Tokyo, Japan). After the abdominal aorta between the left renal artery and iliac bifurcation was carefully exposed, CaCl 2treated gauze was attached to the surface of abdominal aorta for 15 min. The gauze was them removed, and the intraperitoneal cavity was thoroughly washed three times with 0.9% sodium chloride (NaCl). In the sham-operated mice, CaCl 2 was substituted with 0.9% NaCl. For the MKP-1 inhibition experiment, BCI (Dual Specificity Protein Phosphatase 1/6 Inhibitor) (HY-115502; MedChemExpress, Monmouth Junction, NJ, USA) was intraperitonially injected twice (at day 3 and 10 after CaCl 2 application) at the dose of 35 mg/kg in 100 µL of 10%DMSO, 40% PEG300, 5% Tween80, and 45% Saline, as previously described [23].

Hemodynamic Analysis
Under conscious and unrestricted conditions, blood pressure and heart rate were recorded using a sphygmomanometer (BP-98A; Softron, Tokyo, Japan).

Serum Concentration of Corticosterone
Serum corticosterone concentration measurements were sourced from FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan.

Aneurysm Measurement and Histological Analysis
Transcardial perfusion with 4% paraformaldehyde was performed under anesthesia. The abdominal aortic tissue was detached by removing the adjacent fatty and scar tissues to ensure that the aortic wall clearly discernible. After recording images, we measured the maximal diameter of aneurysmal aortas using ImageJ software v1.50i (https://imagej.nih. gov/ij/index.html, accessed on 20 November 2021). The abdominal aorta, including a part of the maximal diameter, was cut out and embedded in paraffin. Cross-sections of the aortic tissue were stained with Elastica van Gieson or Masson's trichrome stain. The aneurysmal and non-aneurysmal portions of the aortic sections were defined according to the structural integrity of an elastic plate in Elastica van Gieson or Masson's trichrome stain.

Immunohistochemical Analysis
Three consecutive sections were prepared from the middle portion of the maximal diameter of the AAA. For the F4/80 immunological staining, the anti-F4/80 antibody (1:100, ab6640; Abcam, Cambridge, UK) and Alexa Fluor 488-conjugated secondary antibody (Thermo Fisher Scientific, Waltham, MA, USA) were used. For the matrix metalloproteinase (MMP)-9 staining, the anti MMP-9 antibody (1:100, ab38898; Abcam) and Alexa Fluor 555-conjugated secondary antibody (Thermo Fisher Scientific) were used. The anti-α-SMA antibody (1:200, ab5694; Abcam), Alexa Fluor 488-conjugated secondary antibody (Thermo Fisher Scientific), and Alexa Fluor 555-conjugated secondary antibody (Thermo Fisher Scientific) were used. For MKP-1, the anti-MKP-1 antibody (1:200, sc-373841; Santa Cruz Biotechnology, Dallas, TX, USA) and Alexa Fluor 488-conjugated secondary antibody (Thermo Fisher Scientific) were used. The sections were examined using an LSM 510 META confocal microscope (Carl Zeiss, Jena, Germany). Non-immune immunoglobulin Rabbit IgG and polyclonal isotype control were used as negative controls for α-SMA and MMP-9. For the non-immune immunoglobulin Rat IgG2b, the kappa monoclonal isotype control was used as the negative control for F4/80, and for the nonimmune immunoglobulin mouse IgG2b, the kappa monoclonal isotype control was used as the negative control for MKP-1. Positive staining was analyzed using ImageJ software v1.50i (https://imagej.nih. gov/ij/index.html, accessed on 20 November 2021). The number of F4/80-and MMP-9positive stained nuclei were assessed in two sections comprising six animals from each group. The percentages of the MKP-1-positive stained nuclei were assessed in two sections comprising six animals from the control group and eight animals from the defeat group. The percentages of the MKP-1-positive stained nuclei in the α-SMA-positive stained nuclei were assessed in two sections comprising four animals form the control group and four animals from the defeat group.

Quantitative Real-Time Polymerase Chain Reaction (qPCR)
The total RNA content was extracted from the abdominal aortic tissue using the RNeasy Fibrous Tissue Mini Kit (74704; Qiagen, Hilden, Germany) and was reverse transcribed to prepare cDNA with the TAKARA Prime Script RT reagent Kit with gDNA Eraser (RR047A; Takara Bio, Shiga, Japan). Real-time PCR was performed using a CFX384 Touch Real-Time PCR System (Bio-Rad Laboratories, Inc., Hercules, CA, USA) with a KAPA SYBR ® FAST Universal qPCR Kit (KK4602; KAPA Biosystems, Wilmington, MA, USA). The data are shown as gene expression levels relative to those of the controls. The primer pairs are listed in the Supplementary Table S1.

Ex Vivo MMP Activity
Ex vivo matrix metalloproteinase (MMP) activity was examined using an in vivo imaging system (IVIS), as previously described [24]. An amount of 2 nmol MMPSense 750 FAST (Perkin Elmer, Boston, MA, USA) was injected via the tail vein. The abdominal aorta was excised 6 h after injection, followed by ex vivo aorta imaging using an IVIS Lumina Series III optical imaging platform (PerkinElmer Inc.) while adjusting the red filter (excitation, 749 nm; emission, 775 nm long pass). Regions of interest (ROIs) encircling the abdominal aorta were drawn by hand, and the certifying signal was evaluated in units of scaled counts per second.

Primary Culture of VSMCs in the Thoracic Aorta
The vascular smooth muscle cells (VSMCs) of the descending thoracic aorta were prepared from the 8-week-old control and defeated mice as previously described [25] because the amount of tissue in the infrarenal aorta was much smaller than the amount in the thoracic aorta. The media was excised from the descending thoracic aorta, followed by incubation with 1 mg/mL collagenase type II (Worthington Biochemical Corporation. Lakewood, NJ, USA) to remove endothelial and adventitial cells. The aortic medias were spread in medium containing 1 mg/mL collagenase type II, 0.5 mg/mL elastase type III (Sigma-Aldrich, St. Louis, MO, USA), and 20% fetal bovine serum (FBS). Cell suspensions were centrifuged at 500× g for 5 min, and the cell pellets were resuspended in Dulbecco's modified eagle medium (DMEM) containing 100 U/mL penicillin, 100 µg/mL streptomycin, and 20% FBS. VSMCs were stimulated with 20 ng/mL PDGF-BB (PMG0045; Thermo Fisher Scientific) or 10 ng/mL TGF-β (766-MB; R&D Systems, Minneapolis, MN, USA), as shown in each experiment.

BrdU Assay
The BrdU assay was performed using the 5-bromodeoxyuridine (BrdU) ELISA Kit (ab126556, Abcam). Briefly, the cells were seeded in a 96-well plate at 5 × 10 3 cells per well and were stimulated with 20 ng/mL PDGF (Thermo Fisher Scientific) for 24 h under starvation conditions. The cells were incubated with BrdU reagent for 6 h before the end of PDGF stimulation. After incubation with fixing solution, the anti-BrdU monoclonal antibody was added for 60 min at room temperature, followed by incubation with peroxidase goat anti-mouse IgG conjugate. The cells were then incubated with tetraliethylbenzidine peroxidase substrate for 30 min. After terminating the reaction by adding the stop solution, the absorbance was determined at 450 nm using a Tecan Infinite m200 plate reader.

Statistical Analysis
Data are expressed as the mean ± standard error of the mean (SEM). After examining the normality of distribution and equal variances, a Student's t-test or analysis of variance (ANOVA) was used to analyze significant differences between the groups, followed by the Tukey-Kramer test. For the dependent variables: a non-aneurysmal portion vs. aneurysmal portion, PDGF stimulation, and phosphatase inhibitor treatment, significant differences were examined using two-way ANOVA. Statistical significance was set at p < 0.05. All analyses were performed using GraphPad Prism Ver 8.4.3 for Windows OS (GraphPad Software, LLC, San Diego CA, USA).

Development of AAA Is Promoted in Defeated Mice
Eight-week-old male WT mice underwent RSD and were subjected to the behavior analysis test. The total immobility duration was markedly extended in the RSD-exposed mice than it was in the non-exposed mice (Supplementary Figure S1A). After the social interaction test, 28 of the 47 RSD-exposed mice showing an SIR less than 1.0 were certified as defeated mice. On the other hand, five non-exposed mice showing an SIR less than 1.0 were excluded in the following experiments (Supplementary Figure S1B). The body weight and hemodynamic parameters before CaCl 2 application were equivalent between the two groups (Supplementary Figure S2). The serum corticosterone concentrations were also similar between the two groups (Supplementary Figure S3). After CaCl 2 application, the maximum outer diameters of the AAA grew in a time-dependent manner in both the control and defeated mice. However, no discernible differences were detected between Cells 2022, 11, 732 6 of 21 the two groups ( Figure 1A,B). On the other hand, the circumferences of the external elastic membranes increased significantly in the defeated mice compared to those in the control mice 2 weeks after CaCl 2 application ( Figure 1C,D), suggesting that RSD promoted AAA expansion in the early phases of development.
weight and hemodynamic parameters before CaCl2 application were equivalent between the two groups (Supplementary Figure S2). The serum corticosterone concentrations were also similar between the two groups (Supplementary Figure S3). After CaCl2 application, the maximum outer diameters of the AAA grew in a time-dependent manner in both the control and defeated mice. However, no discernible differences were detected between the two groups ( Figure 1A,B). On the other hand, the circumferences of the external elastic membranes increased significantly in the defeated mice compared to those in the control mice 2 weeks after CaCl2 application ( Figure 1C,D), suggesting that RSD promoted AAA expansion in the early phases of development. Values represent the mean ± SEM for four control and three defeated mice at 1 week, six control and for eight defeated mice at 2 weeks, and seven control and seven defeated mice at 4 weeks. ** p < 0.01 vs. control at 1 and 2 weeks after CaCl2 application. ## p < 0.01 vs. defeat at 1 and 2 weeks after CaCl2 application; two-way repeated measures ANOVA with the Tukey-Kramer post hoc test. (C,D) Representative photographs of Elastica van Gieson stain and quantitative analysis of the circumferences of the external elastic membranes at 1, 2, and 4 weeks after CaCl2 application. Scale bar = 200 μm. Values represent the mean ± SEM for four control and three defeated mice at 1 week, six control and eight defeated mice at 2 weeks, and seven control and seven defeated mice at 4 weeks. * p < 0.05 vs. control at 1 week after CaCl2 application. # p < 0.05 vs. defeat at 1 week after CaCl2 application. ¶ p < 0.05 vs. control at 2 Values represent the mean ± SEM for four control and three defeated mice at 1 week, six control and for eight defeated mice at 2 weeks, and seven control and seven defeated mice at 4 weeks. ** p < 0.01 vs. control at 1 and 2 weeks after CaCl 2 application. ## p < 0.01 vs. defeat at 1 and 2 weeks after CaCl 2 application; two-way repeated measures ANOVA with the Tukey-Kramer post hoc test. (C,D) Representative photographs of Elastica van Gieson stain and quantitative analysis of the circumferences of the external elastic membranes at 1, 2, and 4 weeks after CaCl 2 application. Scale bar = 200 µm. Values represent the mean ± SEM for four control and three defeated mice at 1 week, six control and eight defeated mice at 2 weeks, and seven control and seven defeated mice at 4 weeks. * p < 0.05 vs. control at 1 week after CaCl 2 application. # p < 0.05 vs. defeat at 1 week after CaCl 2 application. ¶ p < 0.05 vs. control at 2 weeks after CaCl 2 application; two-way repeated measures ANOVA with the Tukey-Kramer post hoc test.

Repeated Social Defeat Does Not Enhance the Acute Inflammatory Response after CaCl 2 Application
We first examined the acute inflammatory response 1 week after CaCl 2 application. Immunohistochemical staining for F4/80 and MMP-9 did not show any discernible differences between the two groups ( Figure 2A,B). Consistently, the mRNA expression levels of the inflammatory cytokines were comparable between the two groups ( Figure 2C). We further examined the MMP activity using ex vivo imaging 1 week after CaCl 2 application; however, there were no discernable differences between the two groups ( Figure 2D,E), suggesting that the acute inflammatory response after CaCl 2 application was equivalent between the two groups and that it was not likely to contribute to augmented AAA expansion in defeated mice.

Repeated Social Defeat Inhibits Perivascular Fibrotic Healing after the Acute Inflammatory Response
We focused on fibrotic wound healing after the acute inflammatory response to clarify the augmented AAA expansion mechanism in defeated mice. At 1 and 4 weeks after CaCl 2 application, there no significant differences were observed in the fibrotic areas between the two groups ( Figure 3A,B). In contrast, the fibrotic area in the aneurysmal portion was significantly reduced in the defeated mice compared to in the control mice 2 weeks after CaCl 2 application, whereas no significant differences were observed between the two groups in the non-aneurysmal portion ( Figure 3C,D). We further examined the α-SMA-positive areas in the tunica media and adventitia ( Figure 3E). In the tunica media, the α-SMA-positive areas in the aneurysmal lesions were scarcely observed in both the control and defeated mice ( Figure 3F). However, in the adventitia, the α-SMA-positive areas in aneurysmal lesions were markedly decreased in the defeated mice compared to in the control mice, whereas there were no discernable differences observed in the nonaneurysmal portions between the two groups ( Figure 3G). These findings suggest that fibrotic wound healing was inhibited in the defeated mice, independent of the reduction in the α-SMA-positive areas in the tunica media.

Repeated Social Defeat Does Not Enhance the Acute Inflammatory Response after CaCl2 Application
We first examined the acute inflammatory response 1 week after CaCl2 applicatio Immunohistochemical staining for F4/80 and MMP-9 did not show any discernible diffe ences between the two groups ( Figure 2A,B). Consistently, the mRNA expression leve of the inflammatory cytokines were comparable between the two groups ( Figure 2C). W further examined the MMP activity using ex vivo imaging 1 week after CaCl2 applicatio however, there were no discernable differences between the two groups ( Figure 2D,E suggesting that the acute inflammatory response after CaCl2 application was equivale between the two groups and that it was not likely to contribute to augmented AAA e pansion in defeated mice.

Repeated Social Defeat Inhibits Perivascular Fibrotic Healing after the Acute Inflammatory Response
We focused on fibrotic wound healing after the acute inflammatory response to clarify the augmented AAA expansion mechanism in defeated mice. At 1 and 4 weeks after

PDGF-Induced ERK Phosphorylation and BrdU Incorporation in VSMCs Are Inhibited in Defeated Mice
To examine the mechanisms of impaired fibrotic healing in defeated mice, we cultured VSMCs from the thoracic aorta of control and defeated mice. There were no discernible differences in proliferation under the conventional culture conditions. However, the ERK phosphorylation after PDGF stimulation was significantly lower in the VSMCs of defeated mice than it was in the control mice ( Figure 4A,B). Consistently, PDGF-induced BrdU uptake in the VSMCs of defeated mice was significantly lower than it was in control mice ( Figure 4C). We examined the phosphorylation of JNK-1 after PDGF stimulation; however, no differences were observed between the two groups (Supplementary Figure S4A,B). Furthermore, Smad-2 phosphorylation after TGF-β stimulation was comparable between the two groups (Supplementary Figure S5A,B). These findings suggest that the impaired VSMC proliferation in the defeated mice is implicated in the PDGF-activated ERK signaling pathway.
1, x FOR PEER REVIEW 9 of 21 the α-SMA-positive areas in the aneurysmal lesions were scarcely observed in both the control and defeated mice ( Figure 3F). However, in the adventitia, the α-SMA-positive areas in aneurysmal lesions were markedly decreased in the defeated mice compared to in the control mice, whereas there were no discernable differences observed in the nonaneurysmal portions between the two groups ( Figure 3G). These findings suggest that fibrotic wound healing was inhibited in the defeated mice, independent of the reduction in the α-SMA-positive areas in the tunica media.   mice ( Figure 4C). We examined the phosphorylation of JNK-1 after PDGF stimulation; however, no differences were observed between the two groups (Supplementary Figure  S4A,B). Furthermore, Smad-2 phosphorylation after TGF-β stimulation was comparable between the two groups (Supplementary Figure S5A,B). These findings suggest that the impaired VSMC proliferation in the defeated mice is implicated in the PDGF-activated ERK signaling pathway.

PDGF-Induced MKP-1 Expression in VSMCs Is Augmented in Defeated Mice
To clarify the molecular mechanisms involved in the decreased ERK phosphorylation after PDGF stimulation, we examined the upstream ERK phosphorylation pathways. However, the PDGFRB and Raf phosphorylation were comparable between the two groups (Supplementary Figure S6A,B). We next examined the protein expression levels of MKP-1, which is a negative regulator of MAPK. We first examined the baseline MKP-1 protein expression levels in VSMC without PDGF stimulation; however, MKP-1 did not show high levels of expression in either of the VSMC groups (Supplementary Figure S7). MKP-1 expression after PDGF stimulation was significantly higher in the VSMCs of defeated mice than it was in the control mice ( Figure 5A,B). The MKP-1protein expression level was dependent on the stability that it gained via ERK-mediated phosphorylation [26]. Consistently, the PDGF-induced MKP-1 phosphorylation was markedly increased in the VSMCs of the defeated mice compared to in the control mice ( Figure 5C,D), whereas the PDGF-induced mRNA expression level of MKP-1 was equivalent between the two groups ( Figure 5E). Finally, we examined the effect of the phosphatase inhibitor BCI on impaired ERK phosphorylation in the VSMCs of the defeated mice. After treatment with BCI, the PDGF-induced ERK phosphorylation was comparable between the two groups ( Figure 5F,G). These findings further suggest that the PDGF-induced ERK phosphorylation in the VSMCs of the defeated mice is eliminated by the augmented MKP-1 expression.

MKP-1 Expression in AAA Is Exaggerated in Defeated Mice
We examined MKP-1 expression in AAA and found that MKP-1 mRNA expression was significantly higher in defeated mice than in control mice ( Figure 6A). Consistently, the percentage of MKP-1-positive nuclei was markedly higher in the defeated mice than in the control mice ( Figure 6B,C). Furthermore, the double staining of MKP-1 and α-SMA revealed that the percentage of the MKP-1-positive stained nuclei in the α-SMA-positive cells was markedly higher in the defeated mice than in the control mice ( Figure 6D,E). These findings indicate that the impaired accumulation of α-SMA-positive cells and reduced fibrotic response in the defeated mice was mediated by augmented MKP-1 expression.

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To clarify the molecular mechanisms involved in the decreased ERK phosphorylation after PDGF stimulation, we examined the upstream ERK phosphorylation pathways. However, the PDGFRB and Raf phosphorylation were comparable between the two groups (Supplementary Figure S6A,B). We next examined the protein expression levels of MKP-1, which is a negative regulator of MAPK. We first examined the baseline MKP-1 protein expression levels in VSMC without PDGF stimulation; however, MKP-1 did not show high levels of expression in either of the VSMC groups (Supplementary Figure S7). MKP-1 expression after PDGF stimulation was significantly higher in the VSMCs of defeated mice than it was in the control mice ( Figure 5A,B). The MKP-1protein expression level was dependent on the stability that it gained via ERK-mediated phosphorylation [26]. Consistently, the PDGF-induced MKP-1 phosphorylation was markedly increased in the VSMCs of the defeated mice compared to in the control mice ( Figure 5C,D), whereas the PDGF-induced mRNA expression level of MKP-1 was equivalent between the two groups ( Figure 5E). Finally, we examined the effect of the phosphatase inhibitor BCI on impaired ERK phosphorylation in the VSMCs of the defeated mice. After treatment with BCI, the PDGF-induced ERK phosphorylation was comparable between the two groups ( Figure 5F,G). These findings further suggest that the PDGF-induced ERK phosphorylation in the VSMCs of the defeated mice is eliminated by the augmented MKP-1 expression.

MKP-1 Expression in AAA Is Exaggerated in Defeated Mice
We examined MKP-1 expression in AAA and found that MKP-1 mRNA expression was significantly higher in defeated mice than in control mice ( Figure 6A). Consistently, the percentage of MKP-1-positive nuclei was markedly higher in the defeated mice than in the control mice ( Figure 6B,C). Furthermore, the double staining of MKP-1 and α-SMA revealed that the percentage of the MKP-1-positive stained nuclei in the α-SMA-positive cells was markedly higher in the defeated mice than in the control mice ( Figure 6D,E). These findings indicate that the impaired accumulation of α-SMA-positive cells and reduced fibrotic response in the defeated mice was mediated by augmented MKP-1 expression.

Treatment with MKP-1 Inhibitor Attenuated Exaggerated AAA Development in Defeated Mice
To examine the effect of MKP-1 on the exaggerated aneurysmal expansion in defeated mice, an MKP-1 inhibitor, BCI, was administered after CaCl 2 application. No significant differences were observed in neither the maximum outer diameters nor the circumferences of the external elastic membrane between the two groups of BCI-treated mice ( Figure 7A-D). The perivascular fibrotic area was also comparable between the two groups ( Figure 7E,F). These findings support the notion that augmented MKP-1 expression contributes to the attenuated early fibrotic response along with the exaggerated expansion of AAA in defeated mice.

MKP-1 Expression in AAA Is Exaggerated in Defeated Mice
We examined MKP-1 expression in AAA and found that MKP-1 mRNA expression was significantly higher in defeated mice than in control mice ( Figure 6A). Consistently, the percentage of MKP-1-positive nuclei was markedly higher in the defeated mice than in the control mice ( Figure 6B,C). Furthermore, the double staining of MKP-1 and α-SMA revealed that the percentage of the MKP-1-positive stained nuclei in the α-SMA-positive cells was markedly higher in the defeated mice than in the control mice ( Figure 6D,E). These findings indicate that the impaired accumulation of α-SMA-positive cells and reduced fibrotic response in the defeated mice was mediated by augmented MKP-1 expression.

Treatment with MKP-1 Inhibitor Attenuated Exaggerated AAA Development in Defeated Mice
To examine the effect of MKP-1 on the exaggerated aneurysmal expansion in defeated mice, an MKP-1 inhibitor, BCI, was administered after CaCl2 application. No significant differences were observed in neither the maximum outer diameters nor the circumferences of the external elastic membrane between the two groups of BCI-treated mice ( Figure 7A-D). The perivascular fibrotic area was also comparable between the two groups ( Figure 7E,F). These findings support the notion that augmented MKP-1 expression contributes to the attenuated early fibrotic response along with the exaggerated expansion of AAA in defeated mice.

Discussion
We showed that RSD enhances CaCl 2 -induced AAA development and impaired fibrotic wound healing following an acute inflammatory response. Consistently, the PDGFinduced ERK phosphorylation and subsequent DNA synthesis in the primarily cultured VSMCs from the defeated mice was significantly reduced compared to in the control mice. The PDGF-induced MKP-1 protein expression was noticeably higher in the VSMCs from the defeated mice than in it was in the control mice, and treatment with the phosphatase inhibitor completely restored ERK phosphorylation to the same extent as seen in the control mice. Finally, the mRNA and protein expression of MKP-1 in AAA was markedly enhanced by RSD. These findings suggest that RSD exaggerates AAA development by eliminating fibrotic wound healing via the MAPK-MKP-1 signaling pathway, indicating that the restoration of the wound healing process could be a potential therapeutic target in depression-related AAA expansion.
Maegdefessel et al. demonstrated that an early fibrotic response in the abdominal aortic wall plays a crucial role in inhibiting AAA progression in gene-manipulated mice [27]. The aortic expression of miR-29b, a known target for collagen-synthesis genes, was significantly decreased in both the porcine pancreatic elastase infusion model and in the Angiotensin II infusion model along with AAA development. Treatment with anti-miR-29b treatment significantly augmented collagen deposition, leading to a reduction in AAA development, and vice versa, as well as an overexpression of miR-29b. Lindeman et al. also demonstrated that the adventitial collagen fibers that encage the vessel prevent the vessel from overstretching and that their functional failure is associated with the expansion of the abdominal aneurysm in patients with AAA [28]. These findings suggest that adventitial fibrosis following acute inflammation plays an important role in preventing the progression of AAA in humans as well as in animal models.
Cole-King et al. were the first to report that the severity of depression was significantly correlated with delayed wound healing after punch biopsy [29]. Numerous human studies have shown that the patients who experience the highest levels of depression and anxiety are more likely to exhibit impaired wound healing after surgery [30,31]. In animal studies using various types of wound healing models, psychological stress also delayed the wound healing process, implicating the inflammatory cytokines and hypothalamic-pituitaryadrenal (HPA) activity [30]. Fibrosis is a multifactorial process, and macrophage-mediated inflammatory responses contribute to subsequent inflammation-associated fibrosis [32]. Indeed, Goldberg et al. showed that inflammatory cytokines such as TNF-α can inhibit myofibroblast differentiation and can contribute to a delay in the wound healing process [33]. However, given that the mRNA expression of inflammatory cytokines and the serum concentration of corticosterone were not affected by RSD, it is likely that the impaired wound healing observed in defeated mice is independent of the acute inflammatory response.
Duric et al. showed a significant increase in the expression of MKP-1, a negative regulator of ERK phosphorylation, in the hippocampal subfields of postmortem tissue from subjects with major depressive disorder (MDD) [34]. They also demonstrated that the hippocampal expression of MKP-1 was increased in rat and mouse models of depression. Likewise, Wang et al. showed that ERK1/2 activity was significantly downregulated in the prefrontal cortex and hippocampus and that it was accompanied by enhanced MKP activity in patients with depression and in animal models of depression [35]. These findings suggest that the MAPK-MKP-1 signaling pathway in the central nervous system (CNS) plays a crucial role in the pathogenesis of depression. The MAPK-MKP-1 pathway plays a crucial role in VSMC proliferation and migration during vascular remodeling, including in the fibrotic response after arterial injury [36,37]. The phenotypic modulation of VSMCs via the MAPK-MKP-1 signaling pathway is likely to be responsible for the impaired fibrotic response in defeated mice, which was also observed in the neural cells in the CNS of patients with depression.
Because the genes and proteins comprising MKP-1 have a very short half-life, MKP-1 activity is largely dependent on protein stability [38,39]. MKP-1 stability and activity are augmented by the direct phosphorylation of serine 359 and serine 364 through their interaction with ERK1/2, which exerts a well-defined negative-feedback MAPK control mechanism [38]. MKP-1 was highly expressed in the VSMCs of arteries in vivo [40]. The proliferation and migration of the VSMCs following vascular injury may be attributed, at least in part, to a decrease in MKP-1 expression [41]. Based on our findings that treatment with a phosphatase inhibitor completely restored the ERK phosphorylation that had been in the VSMCs from defeated mice and that it augmented MKP-1 gene and protein expression in the perivascular area after CaCl 2 application in the defeated mice, it is likely that the RSDinduced modulation of the MAPK-MKP-1 pathway in VSMCs is implicated in the impaired migration and replication of α-SMA-positive cells, thereby contributing to the decreased fibrotic wound healing process and subsequent AAA expansion in defeated mice.
Epigenetic changes that are modulated by microRNAs (miRNAs) have been intensively explored in patients with major depressive disorder (MDD) and in patients with AAA. Maegdefessel et al. showed that miR29-b is profoundly implicated in AAA expansion in human and animal models [27], and Wan et al. examined differential miRNA expression in the cerebrospinal fluid (CSF) of patients with MDD [42]. They showed that the miR-29b-3p expression levels in the CSF were significantly higher in depressed patients than they were in the control subjects, suggesting the involvement of increased miR-29b expression in depression-related AAA development. Compared to miRNA-29b, the miRNA-146a expression level in patients with MMD has been studied more extensively [43,44]. Hung et al. showed that the miRNA-146a levels in the peripheral blood monocytes from MDD patients were significantly lower than they were in healthy controls, which was partially restored after antidepressant treatment [45]. They focused on the role of miRNA-146b in the inflammatory response via the TLR-4 pathway that promotes the excessive production of pro-inflammatory cytokines. In contrast, Lopez et al. showed that miRNA-146a expression was upregulated in brain tissue from MDD patients who had committed suicide [46]. They also observed a significant downregulation in miR-146a-5p expression in blood samples from depressed patients who responded to antidepressant treatment. Furthermore, they revealed the expression levels of the miR-146a-5p-regulated genes involved in the MAPK pathway. Given that miRNA-146a expression was upregulated in AAA tissue samples from patients [47], augmented miRNA-146a expression might lead to the dysregulation of the MAPK-MKP-1 signaling pathway that impairs the fibrotic response as well as the depression-like behavior observed in defeated mice. ERK1/2 activation has been shown to promote the development of aortic aneurysm in various experimental animal models. Holm et al. [48] reported that TGF-β-induced ERK1/2 activation promoted aortic root dilatation in a mouse model of Marfan syndrome. Ghosh et al. [49] also demonstrated that ERK1/2 activation exaggerated aneurysmal dilatation in a murine elastase infusion model by enhancing MMP activity. Recently, Peng et al. [50] reported that ERK1/2 activation is involved in the VSMC phenotype switching from a contractile to a synthetic phenotype, leading to an increase in proliferation, migration, and MMP activity. However, several kinds of inflammatory cytokines and growth factors are involved in ERK1/2 activation through different receptors and subsequent signal transduction pathways. The intensity and duration of ERK1/2 activation are ligandspecific and are able to determine cellular responses in a cell-specific manner [51]. From this point of view, ERK1/2 activation that is elicited by noncanonical TGF-β signaling or proinflammatory cytokines is preferentially involved in MMP secretions, thereby promoting aortic aneurysm in conventional experimental animal models. In contrast, our findings suggest that PDGF-stimulated ERK1/2 activation is specifically implicated in reducing depression-related AAA development.
The present study has methodological implications. First, we retrieved the primary cultured VSMCs from murine thoracic aortic tissues, but not from the infrarenal aorta. Phenotypic variations between the VSMCs from the thoracic aorta and abdominal aorta have long been reported in terms of embryological origins and subsequent transcriptomic profiles. However, there was less tissue in the infrarenal aorta than there was in the thoracic aorta. Therefore, we examined the primary cultured VSMCs from the descending thoracic aorta, the characterization of which has been well established. Second, adventitial fibroblasts play a crucial role in vascular remodeling [52]. We first focused on the adventitial fibroblasts in the descending thoracic aorta. However, the flow cytometric analysis and the sorting of the adventitial fibroblasts were technically difficult due to the small amount of tissue that could be collected from the descending thoracic aorta. We therefore examined the VSMCs instead of the adventitial fibroblasts. Furthermore, because MKP-1 is a negative regulator of JNK and p-38 as well as of ERK, we cannot exclude the possibility that the augmented MKP-1 expression affected JNK, p-38, ERK phosphorylation in the in vivo experiment. Finally, further phenotypic characterizations of the VSMCs and adventitial fibroblasts, including their migration response and collagen deposition, as well as the involvement of canonical TGF-β signaling need to be investigated in future studies using Cre-driver defeated mice.
We focused on the link between psychological stress and AAA development in the context of wound healing after an inflammatory response. We showed that psychological stress could promote AAA expansion, at least in part, by eliminating perivascular fibrosis through the MAPK-MKP-1 signaling pathway, as is the case for neuronal activity in the brain tissue of patients with MDD. Our findings provide novel insight into the mechanisms of depression-related AAA development, showing that the MAPK-MKP-1 pathway that is involved in the wound healing process can be a potential therapeutic target in depressionrelated AAA.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/cells11040732/s1, Figure S1: Analysis of depression-like behavior, Figure S2: Body weight and hemodynamic parameters before CaCl 2 application were comparable between the two groups, Figure S3: Serum concentrations of corticosterone, Figure S4: Repeated social defeat does not affect PDGF-induced JNK phosphorylation in primary cultured VSMCs, Figure S5: Repeated social defeat does not affect TGF-β-induced Smad2 phosphorylation in primary cultured VSMCs, Figure S6: Repeated social defeat does not affect PDGF-induced PDGFRB and Raf phosphorylation in primary cultured VSMCs, Figure S7: MKP-1 protein expression before PDGF stimulation. Representative Western blot of MKP-1 in VSMCs before and after PDGF stimulation, Table S1: List of primers used in qPCR assay.