SGK1-Mediated Vascular Smooth Muscle Cell Phenotypic Transformation Promotes Thoracic Aortic Dissection Progression

Abstract

BACKGROUND:

The occurrence of thoracic aortic dissection (TAD) is closely related to the transformation of vascular smooth muscle cells (VSMCs) from a contractile to a synthetic phenotype. The role of SGK1 (serum- and glucocorticoid-regulated kinase 1) in VSMC phenotypic transformation and TAD occurrence is unclear.

METHODS:

Four-week-old male Sgk1^F/F (Sgk1 floxed) and Sgk1^F/F;Tagln^Cre (smooth muscle cell–specific Sgk1 knockout) mice were administered β-aminopropionitrile monofumarate for 4 weeks to model TAD. The SGK1 inhibitor GSK650394 was administered daily via intraperitoneal injection to treat the mouse model of TAD. Immunopurification and mass spectrometry were used to identify proteins that interact with SGK1. Immunoprecipitation, immunofluorescence colocalization, and GST (glutathione S-transferase) pull-down were used to detect molecular interactions between SGK1 and SIRT6 (sirtuin 6). RNA-sequencing analysis was performed to evaluate changes in the SIRT6 transcriptome. Quantitative chromatin immunoprecipitation was used to determine the target genes regulated by SIRT6. Functional experiments were also conducted to investigate the role of SGK1-SIRT6-MMP9 (matrix metalloproteinase 9) in VSMC phenotypic transformation. The effect of SGK1 regulation on target genes was evaluated in human and mouse TAD samples.

RESULTS:

Sgk1^F/F;Tagln^Cre or pharmacological blockade of Sgk1 inhibited the formation and rupture of β-aminopropionitrile monofumarate–induced TADs in mice and reduced the degradation of the ECM (extracellular matrix) in vessels. Mechanistically, SGK1 promoted the ubiquitination and degradation of SIRT6 by phosphorylating SIRT6 at Ser338, thereby reducing the expression of the SIRT6 protein. Furthermore, SIRT6 transcriptionally inhibits the expression of MMP9 through epigenetic modification, forming the SGK1-SIRT6-MMP9 regulatory axis, which participates in the ECM signaling pathway. Additionally, our data showed that the lack of SGK1-mediated inhibition of ECM degradation and VSMC phenotypic transformation is partially dependent on the regulatory effect of SIRT6-MMP9.

CONCLUSIONS:

These findings highlight the key role of SGK1 in the pathogenesis of TAD. A lack of SGK1 inhibits VSMC phenotypic transformation by regulating the SIRT6-MMP9 axis, providing insights into potential epigenetic strategies for TAD treatment.

Highlights.

• Loss of vascular smooth muscle cell Sgk1 (serum- and glucocorticoid-regulated kinase 1) or its pharmacological blockade prevented the formation and rupture of β-aminopropionitrile monofumarate–induced thoracic aortic dissections in mice, reducing ECM (extracellular matrix) degradation in blood vessels.

• SGK1 phosphorylates SIRT6 (sirtuin 6) and promotes its ubiquitination and degradation.

• SGK1-SIRT6-MMP9 (matrix metalloproteinase 9) forms a regulatory axis involved in the ECM signaling pathway, regulating vascular smooth muscle cell phenotypic transformation.

• SGK1, as a driver of thoracic aortic dissection progression, is a potential target for future drug development and selective treatment.

Thoracic aortic dissection (TAD) is a severe cardiovascular disease caused by the inability of vessel walls to withstand intraluminal high pressure, leading to expansion of the aortic wall and the formation of an aneurysm, with the inner layer rupturing to form a dissection, ultimately leading to aortic rupture and sudden death.¹ The rapid progression of the disease and the high incidence of the acute phase of this disease are the main clinical features of TAD. The annual incidence of TAD is estimated to be 7.6 cases per 100 000 people, and the mortality rate due to TAD rupture in patients who are not diagnosed and treated in a timely manner is as high as 80%.^2,3 Currently, the understanding of the pathogenesis of TAD is limited, but pathological features, including loss and rupture of elastic fibers, abnormalities in the inner layer cells and ECM (extracellular matrix) of the aortic wall, and loss or proliferation of vascular smooth muscle cells (VSMCs), are known to occur.^4,5 Despite significant improvements in treatment, particularly in surgical methods and techniques, the mortality rate remains high, and effective drug therapies are lacking.

VSMCs are important components of the aorta and exhibit significant phenotypic changes, transforming from a quiescent state in mature arteries a proliferative and synthetic state in the context of disease or injury.⁶ The quiescent state of contractile VSMCs counteracts the progression of various forms of cardiovascular diseases and plays a crucial role in maintaining the normal structure and function of the aorta. Synthetic VSMCs are capable of synthesizing MMPs (matrix metalloproteinases), particularly MMP2 and MMP9, which degrade protein substrates, promoting cell proliferation and migration.⁶ The ECM, which includes collagen, elastin, glycoproteins, and proteoglycans and serves as a substrate for MMP9, is located primarily in the media and adventitia and plays a crucial role in vascular elasticity (compliance) and integrity.⁷ Increasing evidence indicates that the phenotypic transformation of VSMCs plays a key role in the development and rupture of TADs in humans and mice, although the specific underlying mechanisms remain unclear.^8,9

SGK1 (serum- and glucocorticoid-regulated kinase 1) is a member of the serine/threonine protein kinase AGC (serine/threonine protein kinases with a conserved catalytic domain) kinase family that shares significant sequence homology and kinase functions with the AKT (protein kinase B [PKB]) family.^10,11 SGK1 can recognize phosphorylation sites, including serine or threonine residues, in substrate proteins, such as the RXRXX (S/T) sequence motif (where X is any amino acid and R is arginine).¹⁰ SGK1 is involved in regulating various ion channels, membrane transporters, and transcription factors and participates in physiological and pathological processes such as cell growth, proliferation, survival, migration, and apoptosis.^12–14 Increasing evidence suggests that SGK1 plays a role in cardiovascular disease.^15–17 An increase in functional mutations in SGK1 can promote hypertension, insulin resistance, and obesity and play an important role in cardiovascular development.^18,19 In addition, SGK1 is involved in vascular calcification, and various triggering factors induce a sharp increase in SGK1 expression during VSMC osteogenic/chondrogenic transdifferentiation.²⁰ SGK1-mediated activation of the transcription factor nuclear factor-κB in activated B cells has been described as a key signaling event promoting VSMC calcification.²¹ Additionally, SGK1 expression and activity are associated with VSMC migration, and gene knockout of SGK1 can reduce cell migration.²² However, the role of SGK1 in TAD has not yet been determined.

The histone deacetylase SIRT6 (sirtuin 6) participates in transcriptional repression by removing acetyl groups from H3K9 (histone 3 lysine 9), H3K56 (histone 3 lysine 56), and H3K18 (histone 3 lysine 18) and plays an important role in cardiovascular diseases such as myocardial hypertrophy, endothelial dysfunction, and atherosclerosis.²³ SIRT6 is considered an important regulator of senescence.²⁴ Sirt6 knockout mice exhibit a progeroid phenotype, while Sirt6-overexpressing mice exhibit an extended lifespan and resistance to the effects of hypoxia on the heart.^25,26 SIRT6 negatively regulates the formation of unstable atherosclerotic plaques in patients with diabetes.²⁷ SIRT6 protein expression is reduced in plaque-like VSMCs in humans and mice, and SIRT6 maintains telomeres and VSMC lifespan through its deacetylase activity, inhibiting the occurrence of atherosclerosis.²⁸ Recent studies have shown that SIRT6 inhibits the expression of IL (interleukin)-1β through the deacetylation of H3K9 and H3K56, suppressing inflammation and senescence and inhibiting thoracic aortic aneurysm.²⁹

In this study, we investigated the role of SGK1 in TAD using an experimental mouse model and human sporadic TAD samples. Furthermore, we explored the interaction between SGK1 and SIRT6, as well as the mechanism through which SGK1 regulates target genes involved in VSMC phenotypic transformation and ECM degradation. We tested the hypothesis that targeting SGK1 may be a potential therapeutic option for slowing the progression of TAD.

Materials and Methods

Data Availability

The expanded Materials and Methods section is given in the Supplemental Material. The data that support the findings of this study are available from the corresponding author upon request.

Antibodies and Reagents

All the research materials listed in the Methods are included in the Major Resources Table in the Supplemental Material.

Animals

Sgk1^F/F (Sgk1 floxed) mice, which were maintained on a C57BL/6J genetic background, were purchased from GemPharmatech (Nanjing, China). The Sgk1 gene has 13 transcripts. According to the structure of the Sgk1 gene, exons 4 to 14 of the Sgk1-204 (ENSMUST00000120509.7) transcript are recommended as the knockout region. The region contains a 1220-bp coding sequence. Knockout of the region will result in protein function disruption. The Sgk1 gene was used to modify CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat–associated 9) technology in this study. C57BL/6J Tagln;iCre mice were purchased from GemPharmatech (Nanjing, China). The Tagln gene has 3 transcripts. According to the structure of the Tagln gene, Tagln-201 (ENSMUST00000034590.3) was selected for the presentation of the recommended strategy. The Tagln-201 gene has 5 exons, with the ATG (adenine-thymine-guanine) start codon in exon 2 and TAG (thymine-adenine-guanine) stop codon in exon 5. Tagln-iCre-PolyA knock-in mice were made via the CRISPR/Cas9 system.³⁰ Sgk1^F/F mice were hybridized with C57BL/6J Tagln;iCre mice to generate Sgk1^F/F;Tagln^Cre (smooth muscle cell–specific Sgk1 knockout) mice. All animals used for the experiments were male because of their small sex hormone variations and high incidence of TAD. Sgk1^F/F and Sgk1^F/F;Tagln^Cre mice aged 4 weeks were administered β-aminopropionitrile monofumarate (BAPN) in the drinking water (1 g/kg per day; Sigma-Aldrich, St. Louis) for 4 weeks to induce aortic dissection (n=16 per group). Control group without BAPN treatment (n=7 per group). GSK650394 was administered daily via an intraperitoneal injection (20 mg/kg). We used the PROC POWER procedure in the SAS software to estimate the sample size. The significance threshold and power were set at 0.05 and 80% to 90%, respectively. Effect sizes (expected differences and variances) between groups were based on preliminary experiments and related articles. All animals were housed under standard conditions (20–26 °C; relative humidity, 40%–70%; 12-hour light/dark cycle) with an unlimited supply of food (Beijing Keao Xieli Feed Co, Ltd). After 4 weeks, the mice were euthanized via an intraperitoneal injection of 1% pentobarbital sodium. All animal experimental procedures were approved by the Experimental Animal Ethics Committee of Central Hospital affiliated with Shandong First Medical University (approval number: JNCHIACUC2022-33) and conformed with the Guideline for the Care and Use of Laboratory Animals of the National Institutes of Health.

Ultrasound Imaging

Transthoracic B-mode images of the aorta were obtained for animals lightly anesthetized with isoflurane (RWD Life Science Co, Ltd) using a Vevo 3100LT high-frequency ultrasound system (FUJIFILM VisualSonics, Inc). The fur was removed using depilatory cream applied to the chest and stomach. Each mouse was anesthetized with isoflurane (3% induction and 1.5% maintenance) and placed on a heating pad in the supine position to maintain a body temperature of 37 °C and minimize the confounding effects of fluctuating body temperatures. Images of the thoracic and abdominal aortas were obtained. The maximum transverse dimensions of the aortic arch and abdominal aorta were measured in triplicate at the end of systole. The left ventricular ejection fraction percentage and fractional shortening percentage were automatically calculated using Vevo LAB v.3.2.6 (Fujifilm VisualSonics, Inc) in M mode.

Blood Pressure Measurement

Blood pressure was measured in unanesthetized mice using a BP-2010A Smart noninvasive blood pressure monitor (Softron Biotechnology). All parameters were measured at least 3×, and the average values were ultimately used for comparison.

Human Aortic Samples

In this study, we collected aortic tissue samples from patients who underwent ascending aortic replacement surgery and control tissue samples from those who underwent coronary bypass graft surgery. Aortic tissues were obtained from patients who underwent surgical treatment at the Department of Cardiac Surgery, Central Hospital affiliated with Shandong First Medical University, Jinan City, China. Patients with TAD who received surgical treatment were included if medical imaging confirmed TAD. Patients were excluded if they had a family history of genetic disorders (eg, Marfan syndrome), a history of tumors, or other immune disorders. The study was performed according to the principles of the Declaration of Helsinki for investigation with humans. The Medical Ethics Committee of the Central Hospital affiliated with Shandong First Medical University reviewed and approved this study (approval number: 2023-093-01). Aortic tissues were collected from 5 patients with TAD and 5 control patients after informed consent was obtained. Each sample is labeled with a unique identifier to ensure confidentiality and traceability. After collection, the samples are immediately placed in a portable cooler with ice packs to maintain stable temperatures. They are transported to the laboratory within 1 hour and stored at −80 °C to ensure the integrity of the sample. Samples sizes were determined based on the experience/studies needed. Relevant clinical information is provided in Table S1.

Cell Culture and Transfection

After euthanizing the mice, the aorta was detached from the arch to the iliac bifurcation, stripping the surrounding adipose tissue as much as possible. The aortic medium was dissected under a microscope and cut into slices 1 to 2 mm long. Mouse VSMCs were cultured in DMEM (VivaCell, catalog number: C3113-0500) supplemented with 20% fetal bovine serum (Gibco; catalog number: 10100147) and 1% antibiotics (Gibco; catalog number: 15140122).³¹ HEK-293T (human embryonic kidney 293T) cells and MOVAS (mouse aortic vascular smooth muscle) cell lines were obtained from Procell Life Science & Technology. HEK-293T and MOVAS cells were maintained in DMEM (Gibco) supplemented with 10% fetal bovine serum (Gibco) and penicillin/streptomycin (Gibco; 10 000 U/mL). Human aortic smooth muscle cells (SMCs) were purchased from ScienCell Research Laboratories (Carlsbad, CA). The culture dishes were treated with 2 µg/cm² poly-L-lysine (catalog number: 0413; ScienCell) for 12 to 16 hours before cell seeding. Subsequently, the cells were cultured in SMC medium (catalog number: 1101; ScienCell) containing 2% fetal bovine serum (catalog number: 0010; ScienCell) and P/S (penicillin/streptomycin) solution (catalog number: 0503; ScienCell). All cells were maintained in a humidified incubator at 5% CO₂ and 37 °C.

Transfections were performed using Turbofect (Thermo Scientific) or Lipofectamine RNAiMAX reagent (Thermo Scientific) according to the manufacturer’s instructions. Each experiment was performed in triplicate and repeated at least 3×. For RNAi (RNA interference) experiments, at least 3 independent small interfering RNA (siRNA) sequences were tested for each gene; the sequence with the highest efficiency was used. The sequences of the siRNAs used in this study were obtained from GenePharma and are listed in Table S2.

Immunopurification and Mass Spectrometry

HEK-293T cells were transfected with FLAG (a peptide tag consisting of eight amino acids [DYKDDDDK])-tagged SGK1 for 48 hours, and a cell line stably expressing FLAG-SGK1 was obtained. Anti-FLAG immunoaffinity columns were prepared using an anti-FLAG M2 affinity gel (Sigma-Aldrich) according to the manufacturer’s instructions. The FLAG protein complex was eluted from the column using the FLAG peptide (0.2 mg/mL; Sigma-Aldrich). Elution fractions were collected, resolved on SDS-polyacrylamide gels, silver stained, and subjected to liquid chromatography–tandem mass spectrometry and data analysis.

Immunoprecipitation and Western Blotting

For immunoprecipitation assays, cells were washed twice with cold PBS, and extracts were prepared by incubating cells in lysis buffer (50 mmol/L Tris-HCl [pH 7.4], 150 mmol/L NaCl, 2 mmol/L EDTA, 0.3% NP-40, and protease inhibitor cocktail) for 1 hour at 4 °C, followed by centrifugation at 12 000g for 10 minutes. Next, 500 μg protein samples were incubated with the appropriate primary antibodies or normal rabbit/mouse IgG at 4 °C overnight with constant rotation and then mixed with Dynabeads Protein G beads (Invitrogen) for 2 hours at 4 °C. After washing the beads 3× with cell lysis buffer (50 mmol/L Tris-HCl [pH 8.0], 150 mmol/L NaCl, 2 mmol/L EDTA, 0.1% NP-40), the captured immune complexes were separated using 8% to 10% SDS-PAGE with Bis-Tris gels, transferred to polyvinylidene fluoride membranes (Merck Millipore, Billerica, MA), blocked in 5% skim milk, and blotted with secondary antibodies. The stained bands were detected using enhanced chemiluminescence (Merck Millipore) according to the manufacturer’s instructions.

In Vitro Kinase Assay

Recombinant GST (glutathione S-transferase)-SIRT6 was used as a substrate and incubated with recombinant human active SGK1 (Biotechne; 3200-KS-010) in kinase assay buffer containing 25 mmol/L MOPS (3-(N-morpholino) propanesulfonic acid) at pH 7.2, 12.5 mmol/L β-glycerophosphate, 25 mmol/L MgCl₂, 5 mmol/L EGTA, 2 mmol/L EDTA, and 0.25 mmol/L DTT (dithiothreitol) at 30 °C for 30 minutes. The reactions were stopped by adding SDS-PAGE sample buffer. The phosphorylation of SIRT6 was detected by Western blot using anti-phosphoserine antibody.

Immunofluorescence Staining

For immunofluorescence, the cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.2% Triton X-100 in PBS for 10 minutes, and incubated with 0.1% Triton X-100 and 0.4% BSA in PBS at room temperature. The cells were then incubated at 4 °C overnight with primary antibodies. The secondary antibody was incubated with the cells at room temperature for 1 hour. Coverslips were mounted on glass slides using Vectashield with DAPI (4′,6-diamidino-2-phenylindole). Imaging was performed using a TCS SP8 Leica confocal laser scanning microscope, and images were acquired using Leica Application Suite X (Leica, Wetzlar, Germany).

IHC and Histological Analysis

For the IHC assay, paraffin sections of the samples were dewaxed with dimethylbenzene and dehydrated using an alcohol gradient (100%, 90%, and 75%). After antigen retrieval with citrate buffer, the samples were blocked with 3% H₂O₂ and incubated with donkey serum. The slides were incubated with antibodies overnight at 4 °C, followed by incubation with the appropriate HRP (horseradish peroxidase)-conjugated IgG as a polyclonal antibody for 30 minutes at 37 °C. DAB and hematoxylin were obtained from Beijing Zhong Shan Golden Bridge Biological Technology Co, Ltd (Beijing, China) and were used to stain the slides. Imaging was performed using an Olympus-BX530 microscope (Tokyo, Japan).

For histological analysis, paraffin sections of the samples with a thickness of 5 µm were subjected to hematoxylin and eosin (Servicebio), elastic van Gieson (Sigma-Aldrich), and Masson trichrome (Servicebio) staining according to the manufacturer’s instructions. All images were captured using a Pannoramic SCAN II (3DHISTECH, Ltd, Hungary).

GST Pull-Down Experiments

Escherichia coli BL21 cells (Vazyme Biotech Co, Ltd, Nanjing, China) were transformed with GST fusion constructs, and crude bacterial lysates were prepared by sonication in cold PBS supplemented with protease inhibitors. In vitro transcription and translation experiments were performed with rabbit reticulocyte lysate (Promega, Madison, WI). In the GST pull-down assays, ≈10 μg of the appropriate GST fusion proteins were mixed with 5 to 8 μL of in vitro transcribed/translated products and incubated in binding buffer (0.8% BSA in PBS in the presence of a protease inhibitor mixture) at room temperature for 30 minutes. The binding reaction was then added to 30 μL of Glutathione Sepharose 4B beads (GE Healthcare) and mixed at 4 °C for 2 hours. The proteins were subsequently washed 5× with washing buffer, resuspended in 30 μL of 2× SDS-PAGE loading buffer, and resolved on 10% gels. The protein bands were detected with specific antibodies via Western blotting.

Real-Time Quantitative Polymerase Chain Reaction

Total RNA was isolated from the samples using TRIzol reagent (Invitrogen). cDNA was prepared using a RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, MA). SYBR Green Real-Time PCR Master Mix (TOYOBO, Osaka, Japan) was used for polymerase chain reaction amplification, and the thermal cycling protocol was set according to the manufacturer’s instructions: 95 °C for 1 minute and 40 cycles of 95 °C for 15 s, 60 °C (or optimized temperature) for 15 s, and 72 °C for 45 s, followed by melting curve analysis. An ABI QuantStudio Real-Time PCR System (Applied Biosystems) was used for relative quantitation based on SYBR green fluorescence, and the results were obtained using the comparative Ct method (2−ΔΔCt), with GAPDH serving as an internal control. This assay was performed in triplicate. The sequences of all primers used are listed in Table S3.

Cell Migration Assay

Transwell chamber filters (Becton Dickinson) were coated with Matrigel. mVSMCs (primary mouse vascular smooth muscle cells) were then seeded into the upper chamber at a density of 5×10⁴ cells in a volume of 500 mL of serum-free medium, and the bottom of the chamber contained 500 µL of DMEM supplemented with 10% fetal bovine serum. The cells were fixed and stained with crystal violet after they had migrated or invaded for 18 to 24 hours at 37 °C. The cells on the upper side of the membrane were removed using cotton swabs, while those on the other side were counted. Invaded cells were counted using an inverted light microscope (OLYMPUS, Japan).

5-Ethynyl-2′-Deoxyuridine Assays

5-ethynyl-2′-deoxyuridine (EdU) assays were performed using 3 generations of mVSMCs. Briefly, mVSMCs were incubated with EdU in medium (10 μmol/L) for 3 hours. Then, the cells were fixed in 4% paraformaldehyde for 10 minutes, permeabilized with 0.5% Triton X-100, and stained with Alexa Flur 488, according to the manufacturer’s instructions of the Click-iT EdU Alexa Fluor 488 Imaging Kit (Life Technologies). Then, cells were stained with DAPI (Invitrogen). Images were acquired using the OLYMPUS fluorescence microscope.

SA-β-Gal Staining

SA-β-gal (senescence-associated β-galactosidase) staining was performed using 7 to 10 generations of mVSMCs. At the indicated times, cultured mVSMCs were washed with PBS and fixed with staining fixatives for 15 minutes at room temperature. The fixed cells were stained with fresh SA-β-gal staining solution at 37 °C overnight according to the manufacturer’s protocol (G1580; Solarbio Life Science). The cells were washed with PBS, and the blue areas were considered SA-β-gal positive.

Chromatin Immunoprecipitation and Quantitative Chromatin Immunoprecipitation Assays

Chromatin immunoprecipitation assays were performed using mVSMCs as previously described.³² Briefly, 1×10⁷ cells were cross-linked with 1% formaldehyde, sonicated, precleared, and incubated with 2 to 3 µg of antibody per reaction. The complexes were washed with low- and high-salt buffers, after which the DNA was extracted and precipitated. Quantitative chromatin immunoprecipitation (qChIP) was generated using SYBR Green Real-Time PCR Master Mix. The primers used for qChIP are listed in Table S4.

RNA-Sequencing Analysis

siRNA was used to knock down Sirt6 expression in mVSMCs; 3 independent samples and controls were used in these experiments. Total RNA was extracted and purified using oligo(dT)-attached magnetic beads, and RNA-sequencing was conducted on Illumina NovaSeq 6000 (Illumina, San Diego, CA) by Novogeve (Tianjin, China). Genes that were differentially expressed between groups, with a Q value <0.05 and a fold change >1.5, were identified. The data sets supporting this research are available in the National Center for Biotechnology Information Gene Expression Omnibus repository under accession code GSE246209.

Statistical Analysis

Data analysis was performed with the GraphPad Prism 8.0 software (GraphPad Software, Inc, San Diego, CA). We first assessed the normality of data distributions with the Shapiro-Wilk test and checked for equal variances using the F test. For normally distributed data, we compared 2 groups using a 2-tailed unpaired Student t test or applied 1-way or 2-way ANOVA followed by Tukey post hoc test for >2 groups. If data were not normally distributed, we used the nonparametric Mann-Whitney U test for 2-group comparisons. Data were presented as the mean±SD. A P value of <0.05 was considered statistically significant.

Results

VSMC-Specific Sgk1 Knockout Mitigates TAD Development in Mice

To investigate the role of Sgk1 in aortic dissection, in vivo experiments were conducted using a BAPN-induced TAD mouse model. Initially, Sgk1^F/F mice were crossed with Tagln^Cre mice to generate Sgk1^F/F;Tagln^Cre mice. A total of 46 male mice, aged 4 weeks, for both Sgk1^F/F and Sgk1^F/F;Tagln^Cre mice, were studied (control group, without BAPN treatment, n=7 per group; BAPN-treated group, n=16 per group). Western bloting was used to assess the effects of Sgk1 gene-specific deletion in mVSMCs, and the results indicated that Sgk1 was successfully knocked out at the protein level (Figure S1A). As shown in Figure 1A, the weekly body weight fluctuations of the mice were not significant. After 28 days of BAPN administration, 18.75% (n=3) of the Sgk1^F/F mice and 6.25% (n=1) of the Sgk1^F/F;Tagln^Cre mice succumbed to aortic dissection rupture. Aortic dissection formation was observed in 62.5% (n=10) of the Sgk1^F/F mice and 37.5% (n=6) of the Sgk1^F/F;Tagln^Cre mice following BAPN treatment (Figure 1B through 1D). Vascular ultrasound imaging and measurements of the maximum thoracic aorta diameter on day 28 post-modeling indicated that Sgk1^F/F;Tagln^Cre mitigated BAPN-induced aortic dilation compared with that in Sgk1^F/F mice (Figure 1E and 1F). Abdominal aortic ultrasound demonstrated that BAPN induced abdominal aortic expansion in Sgk1^F/F mice compared with control mice, but there was no significant difference in the maximum abdominal aorta diameter between Sgk1^F/F and Sgk1^F/F;Tagln^Cre mice following BAPN administration (Figure 1G and 1H). Changes in heart function were evaluated using M-mode echocardiography in mice after modeling (Figure S1B). Echocardiography revealed that after BAPN induction, Sgk1^F/F mice exhibited a decreased ejection fraction and fractional shortening; these changes were mitigated by VSMC-Sgk1 knockout (Figure S1C and S1D). Additionally, the average blood pressure and pulse pressure did not significantly differ between the groups after BAPN treatment (Figure 1I and 1J).

Figure 1.

Vascular smooth muscle cell–specific Sgk1 (serum- and glucocorticoid-regulated kinase 1) ablation represses β-aminopropionitrile monofumarate (BAPN)–induced thoracic aortic dissection formation and rupture in mice. A through J, Four-week-old Sgk1^F/F (Sgk1 floxed) and Sgk1^F/F;Tagln^Cre (smooth muscle cell–specific Sgk1 knockout) mice were treated with the control or BAPN for 28 days (n=7 for each control group, n=16 for each BAPN group). A, Body weights of the indicated groups (n=7 for each control group, n=13–16 for each BAPN group). B, Incidence of aortic complications. C, Representative macrographs of the aorta. D, Representative microscopy images of the thoracic aorta. Scale bar=1 mm. E, Representative ultrasound images of the thoracic aorta. Scale bar=1 mm. F, Measurements of the maximum aortic diameter (n=7 for each control group, n=13–15 for each BAPN group). G, Representative ultrasound images of the abdominal aorta. Scale bar=1 mm. H, Measurements of the maximum abdominal aortic diameter (n=7 for each control group, n=13–15 for each BAPN group). I, Mean blood pressure (BP; n=7 for each control group, n=13–15 for each BAPN group). J, Pulse pressure (PP; n=7 for each control group, n=13–15 for each BAPN group). K, Representative hematoxylin and eosin (HE), elastic van Gieson (EVG) and Masson staining images of the thoracic aorta. Scale bar=500 µm. Scale bar=100 µm. Data were presented as mean±SD. Statistical analyses were performed via 2-way ANOVA followed by the Tukey post hoc test.

To further observe the pathological changes in the mice with aortic dissection, chest aorta tissues were stained with hematoxylin and eosin, elastic van Gieson stain, and Masson stain. The results showed that VSMC-Sgk1 knockout inhibited BAPN-induced dissection formation, elastic fiber disruption, and vascular abnormalities (Figure 1K). In summary, these results suggest that the absence of Sgk1 in VSMCs inhibits the formation, development, and rupture of BAPN-induced TADs.

Pharmacological Blockade of Sgk1 Protects Mice From BAPN-Induced TAD

The results of the above studies prompted us to explore whether a similar situation would occur in the case of SGK1 inhibition. C57BL/6J mice were intraperitoneally injected with GSK 650394 (20 mg/kg) daily, which served as an SGK1 inhibitor,³³ within BAPN administration throughout the 4 weeks of modeling. The control groups (without BAPN treatment), n=5 per group; the BAPN-treated groups, n=10 per group. As shown in Figure 2A, no significant weekly fluctuations in body weight were observed among the mice. After 28 days of treatment, the control mice did not exhibit aortic dilation or TAD formation. In the BAPN-treated groups, 20% (n=2) of vehicle-treated mice and 10% (n=1) of GSK 650394–treated mice succumbed to aortic dissection rupture. Aortic dissection formation was observed in 60% (n=6) of vehicle-treated mice and 30% (n=3) of GSK 650394–treated mice following BAPN treatment (Figure 2B through 2D). Ultrasound imaging and measurement of the maximum thoracic aortic diameter on day 28 post-modeling indicated that GSK 650394 alleviated BAPN-induced aortic dilation compared with vehicle treatment (Figure 2E and 2F). These results suggest that the SGK1 inhibitor GSK 650394 inhibits the formation, dilation, and rupture of BAPN-induced TADs. Abdominal aortic ultrasound revealed that BAPN treatment induced abdominal aortic dilation compared with controls; however, no significant difference in maximum abdominal aortic diameter was observed between vehicle-treated and GSK 650394–treated mice (Figure 2G and 2H). Hematoxylin and eosin, elastic van Gieson, and Masson staining presented the same pathological changes in accordance with previous results that Sgk1 inhibition rescued elastin disorganization and vessel wall dissection (Figure 2I), which proved the critical role of SGK1 in TAD progress, making GSK 650394 a promising drug for TAD remedy.

Figure 2.

Inhibition of Sgk1 (serum- and glucocorticoid-regulated kinase 1) with specific inhibitor alleviates thoracic aortic dissection (TAD) progression in mice. A through H, Four-week-old C57BL/6 mice were observed with or without GSK 650394 (20 mg/kg per day) within the control or β-aminopropionitrile monofumarate (BAPN) treatment for 28 days (n=5 for each control group, n=10 for each BAPN group). A, Body weights of the indicated groups (n=5 for each control group, n=8–10 for each BAPN group). B, Incidence of aortic complications. C, Representative macrographs of the aorta. D, Representative microscopy images of the thoracic aorta. Scale bar=1 mm. E, Representative ultrasound images of the thoracic aorta. Scale bar=1 mm. F, Measurements of the maximum aortic diameter (n=5 for each control group, n=8–9 for each BAPN group). G, Representative ultrasound images of the abdominal aorta. Scale bar=1 mm. H, Measurements of the maximum abdominal aortic diameter (n=7 for each control group, n=8–9 for each BAPN group). I, Representative hematoxylin and eosin (HE), elastic van Gieson (EVG), and Masson staining images of the thoracic aorta. Scale bar=500 µm. Scale bar=100 µm. Data were presented as the mean±SD. Statistical analyses were performed via 2-way ANOVA followed by the Tukey post hoc test.

SGK1 Regulates Phenotypic Transformation of VSMC and Interacts With SIRT6

Under stress, VSMCs undergo a transformation from a procontractile phenotype to a prosynthetic phenotype, leading to vascular dysfunction and aortic diseases.^34,35 We further investigated the roles of SGK1 in the phenotypic transformation of VSMC. In mVSMCs, silencing Sgk1 via siRNA resulted in an increase in the mRNA expression of the procontractile markers Myh11, Tagln, Acta2, and Cnn1, as detected by real-time quantitative polymerase chain reaction (RT-qPCR; Figure 3A). Western blot analysis revealed that the protein expression levels of the contractile markers Myh11 (myosin heavy chain 11), α-Sma (alpha smooth muscle actin), and Eln (elastin) increased after Sgk1 knockdown, and this change was accompanied by a decrease in the expression of the synthetic markers Mgp (matrix Gla protein) and Opn (osteopontin; Figure 3B). Furthermore, immunofluorescence was used to detect changes in the fluorescence intensity of the contractile markers α-Sma and Tagln in mVSMCs. The results showed a markedly increase in the fluorescence intensity of α-Sma and Tagln after Sgk1 knockdown (Figure 3C and 3D). The transformation of VSMCs from a contractile to a synthetic phenotype is associated with increased cell migration, secretion capacity, and proliferation.⁶ Next, EdU staining revealed a significant decrease in the percentage of EdU-labeled cells after Sgk1 knockdown (Figure 3E). These findings indicate that Sgk1 deficiency inhibits mVSMC proliferation in vitro. It has been reported that aged VSMCs accumulate in mouse and human TADs.^28,29 However, the role of SGK1 in VSMC aging has been less well characterized. To explore the role of SGK1 in VSMC aging, SA-β-gal staining was performed after silencing Sgk1 in mVSMCs. The results revealed a reduction in cellular senescence after Sgk1 knockdown (Figure 3F). Western blot analysis of senescence markers indicated a decrease in the expression of p53, p21, and p16 after Sgk1 knockdown (Figure 3G). In summary, SGK1 deficiency inhibits the transformation of VSMCs from a contractile to a synthetic phenotype and inhibits cell proliferation and senescence.

Figure 3.

SGK1 (serum- and glucocorticoid-regulated kinase 1) regulates contractile-to-synthetic phenotypic transformation of mVSMCs and interacts with SIRT6 (sirtuin 6). A through G, Control or Sgk1 small interfering RNA (siRNA)–transfected vascular smooth muscle cells (VSMCs) after 48 hours. A, Real-time quantitative polymerase chain reaction (RT-qPCR) data showing the relative mRNA expression levels of the indicated genes in Sgk1 knockdown mVSMCs. The mRNA levels were normalized to those of Gapdh. B, Western blot analysis of the indicated proteins in Sgk1 knockdown mVSMCs. β-Actin served as a loading control for Western blotting. C and D, Representative immunofluorescence staining for the contractile markers Tagln (C) and α-Sma (D) in Sgk1 knockdown mVSMCs. nDNA was stained with DAPI (4′,6-diamidino-2-phenylindole). Scale bar=50 µm. E, Sgk1 knockdown mVSMCs were incubated with 5-ethynyl-2′-deoxyuridine (EdU) for 3 hours. A fluorescence microscope was used to detect EdU (left), and the results were statistically analyzed (right). nDNA was stained with DAPI. Scale bar=100 µm. F, Representative images of SA-β-gal (senescence-associated β-galactosidase)–stained Sgk1 knockdown mVSMCs (left) and statistical analysis (right). The green regions are positively stained. Scale bar=200 µm. G, Western blot analysis of senescence markers in Sgk1 knockdown mVSMCs. β-Actin served as a loading control for Western blotting. H, Immunoaffinity purification and mass spectrometry analysis of SGK1-interacting proteins. Whole-cell extracts from HEK-293T cells stably expressing FLAG (vector) or FLAG-SGK1 were immunopurified using anti-FLAG affinity columns and eluted with the FLAG peptide. The eluates were resolved using SDS-PAGE and silver stained. Protein bands were retrieved and analyzed using mass spectrometry. I, Mass spectrometry analysis of SGK1-interacting proteins. J, Western blot analysis of the purified fractions using antibodies against SIRT6. K, Coimmunoprecipitation (Co-IP) assay of endogenous SGK1 and SIRT6 in HEK-293T, MOVAS, and mVSMC cells. L, Immunoprecipitation (IP) assay in HEK-293T cells ectopically expressing the indicated proteins. M, Normally cultured VSMCs were fixed and analyzed by immunofluorescence using antibodies specific for SGK1 and SIRT6. nDNA was stained with DAPI. Scale bar=100 µm. N, Glutathione S-transferase (GST) pull-down assays with bacterially expressed GST-fused proteins and in vitro transcribed/translated proteins. O, Domain architectures of SIRT6. P, Identification of the essential domains required for interaction. Q, In vitro kinase assay using recombinant human active SGK1 and GST-fused SIRT6 as substrates. R, IP analysis of the serine phosphorylation of SIRT6 in the total lysates of vector- and SGK1-S422D–transfected HEK-293T cells. S, Sequence alignment of the SGK1 phosphorylation motif of SIRT6 from various species. T, IP analysis of HEK-293T cells revealed that SGK1 phosphorylates serine in wild-type (WT) SIRT6 but not in SIRT6-S338A. Data were presented as the mean±SD of 3 independent experiments. Statistical analyses were performed via 2-tailed unpaired t test. α-Sma indicates alpha smooth muscle actin; FLAG, a peptide tag consisting of eight amino acids (DYKDDDDK); MOVAS, mouse aortic vascular smooth muscle cell line; and mVSMC, primary mouse smooth muscle cells.

To understand the molecular mechanism of SGK1 action, affinity purification combined with silver staining and mass spectrometry analysis was performed in HEK-293T cells stably expressing FLAG-tagged SGK1. The results indicated that SGK1 not only interacts with several known proteins but also copurifies with various factors involved in epigenetic regulation (Figure 3H and 3I). The detailed results of the mass spectrometry analysis can be found in Table S5. Further confirmation of the interaction between SGK1 and SIRT6 was obtained through Western blot analysis (Figure 3J). To further validate the interaction between SGK1 and SIRT6, coimmunoprecipitation experiments were conducted in HEK-293T, MOVAS, and mVSMC cells, which showed that SGK1 and SIRT6 coimmunoprecipitated (Figure 3K). Coimmunoprecipitation experiments in HEK-293T cells ectopically expressing SGK1 and SIRT6 also confirmed the physical interaction between these proteins (Figure 3L). To better understand the interaction between SGK1 and SIRT6, immunofluorescence analysis of SGK1 and SIRT6 localization in VSMCs was performed, and the results revealed the colocalization of SGK1 and SIRT6 in the nucleus (Figure 3M).

Subsequently, by using GST-fused SIRT6 and in vitro transcription/translation of SGK1 for GST pull-down, we discovered that SGK1 and SIRT6 interacted directly (Figure 3N). In vitro binding between SGK1 and SIRT6 was assessed by designing SIRT6 fragment plasmids for use in GST pull-down experiments (Figure 3O). Furthermore, the C-terminal amino acid residues 273 to 355 of SIRT6 were found to be responsible for binding to SGK1 (Figure 3P). To further investigate the mechanism underlying the interaction between SGK1 and SIRT6, an in vitro kinase assay was performed using recombinant human active SGK1 and purified GST-fused SIRT6 proteins as substrates. The results revealed that SIRT6 can be directly phosphorylated by recombinant, functionally active SGK1 (Figure 3Q). In HEK-293T cells ectopically expressing active SGK1 (S422D) and SIRT6, immunoprecipitation followed by immunoblot analysis indicated that the active SGK1 protein induces serine phosphorylation of SIRT6 (Figure 3R). SGK1 has a kinase domain highly similar to that of AKT; thus, it shares many substrates containing the AGC kinase consensus motif RXRXX (S/T), where R is arginine, X is any amino acid, and (S/T) is a phosphorylatable serine or threonine.³⁶ Ser338 of SIRT6 is highly conserved in mammals (Figure 3S). To test whether SGK1 directly phosphorylates SIRT6 at the Ser338 site, active SGK1 (S422D), wild-type (WT) SIRT6, and the SIRT6 Ser338 mutant (S338A) were ectopically expressed in HEK-293T cells. Immunoblot analysis revealed a significant reduction in serine phosphorylation levels in the SIRT6-S338A mutant, indicating that Ser338 of SIRT6 is the site phosphorylated by SGK1 (Figure 3T). Taken together, these data suggest that SGK1 interacts with SIRT6 and directly phosphorylates SIRT6 at the Ser338 site.

SGK1 Regulates the Ubiquitination and Degradation of the SIRT6 Protein Through Phosphorylation

Given the interaction of SIRT6 with SGK1 and subsequent SIRT6 phosphorylation, we further investigated whether SGK1 regulates the expression of SIRT6. In mVSMCs, Sgk1 was silenced using siRNA, and the expression of Sgk1 and Sirt6 was examined via RT-qPCR (Figure 4A) and Western blotting (Figure 4B). Upon Sgk1 knockdown, the protein levels of Sirt6 increased, despite comparable mRNA levels. Additionally, mVSMCs were treated with EMD 638683 and GSK 650394, small molecule inhibitors of SGK1. EMD 638683 and GSK 650394 inhibited Sgk1 Ser422 phosphorylation in a time-dependent manner and increased SIRT6 protein expression (Figure 4C and 4D). Furthermore, in HEK-293T cells, the expression of constitutively active SGK1 (SGK1-S422D), but not kinase-defective SGK1 (SGK1-S422A), reduced SIRT6 expression (Figure 4E), indicating a negative correlation between SGK1 activation and SIRT6 expression. Immunofluorescence staining revealed a significant increase in Sirt6 fluorescence intensity in Sgk1-knockdown mVSMCs (Figure 4F).

Figure 4.

SGK1 (serum- and glucocorticoid-regulated kinase 1) modulates SIRT6 (sirtuin 6) protein ubiquitination and degradation. A, Real-time quantitative polymerase chain reaction (RT-qPCR) data showing the relative mRNA expression levels of Sgk1 and Sirt6 in control oligonucleotide- or siSgk1-transfected mVSMCs. The mRNA levels were normalized to those of Gapdh. B, Western blot analysis of Sgk1 and Sirt6 in control and siSgk1-infected mVSMCs. β-Actin served as a loading control for Western blotting. C and D, Western blot analysis of Sirt6, pSgk1-Ser⁴²², and Sgk1 in mVSMCs treated with EMD 638683 (50 µmol/L for 0, 1, or 2 hours; C) or GSK 650394 (20 µmol/L for 0, 1, or 2 hours; D), which act as Sgk1 inhibitors. β-Actin served as a loading control for Western blotting. E, Overexpression of active SGK1-S422D decreased SIRT6 protein levels compared with overexpression of inactive SGK1-S422A in HEK-293T cells. β-Actin served as a loading control for Western blotting. F, Representative fluorescence images of Sgk1 and Sirt6 in control- or siSgk1-transfected mVSMCs. nDNA was stained with DAPI (4′,6-diamidino-2-phenylindole). Scale bar=50 µm. G, Overexpression of active SGK1-S422D increased SIRT6 ubiquitination, as determined by Western blotting for HA-Ub (hemagglutinin [HA]-tagged ubiquitin). HEK-293T cells were cotransfected with wild-type (WT) SGK1 (GFP-SGK1), inactive SGK1 (GFP-SGK1-S422A), or active SGK1 (GFP-SGK1-S422D) in combination with FLAG-SIRT6 and HA-ubiquitin (Ub) for 48 hours. Whole-cell extracts from HEK-293T cells stably expressing FLAG-SIRT6 were immunopurified using anti-FLAG affinity columns and eluted with the FLAG peptide. H, Overexpression of active SGK1-S422D increased SIRT6 ubiquitination, as determined by Western blotting for HA-Ub in HEK-293T cells transfected with the indicated plasmids in the presence of MG132 (10 μmol/L for 7 hours). I, Ubiquitination of SIRT6 and SIRT6-S338A with overexpression of active SGK1-S422D by Western blot in HEK-293T cells. J, Overexpression of active SGK1-S422D decreases WT SIRT6 but not SIRT6-S338A protein levels compared with inactive SGK1-S422A overexpression. β-Actin served as a loading control for Western blotting. K, Western blotting in lysates from HEK-293T cells transfected with FLAG-tagged SIRT6 and either vector or GFP-SGK1-S422D in the presence of cycloheximide (CHX) for up to 15 hours. GAPDH served as a loading control for Western blotting. L, Western blotting in lysates from HEK-293T cells transfected with FLAG-tagged SIRT6-S338A and either vector or GFP-SGK1-S422D in the presence of CHX for up to 15 hours. GAPDH served as a loading control for Western blotting. M, Western blot analysis of Sirt6 in control and siMdm2-infected mVSMCs. β-Actin served as a loading control for Western blotting. N, Ubiquitination of SIRT6 and SIRT6-S338A with overexpression of MDM2 by Western blot in HEK-293T cells. O, Overexpression of MDM2 decreases WT SIRT6 but not SIRT6-S338A protein levels. β-Actin served as a loading control for Western blotting. Data were presented as the mean±SD of 3 independent experiments. A, Data were statistically analyzed by 1-way ANOVA followed by Tukey post hoc test. K and L, Data were statistically analyzed by 2-way ANOVA followed by the Tukey post hoc test. FLAG indicates a peptide tag consisting of eight amino acids (DYKDDDDK); MDM2, mouse double minute 2 homolog; mVSMC, primary mouse smooth muscle cells; and siMdm2, small interfering RNA targeting Mdm2.

To further investigate the molecular mechanism by which SGK1 regulates SIRT6 protein expression, ubiquitination experiments were conducted in HEK-293T cells. The expression of constitutively active SGK1 (S422D) resulted in a greater increase in the ubiquitination level of SIRT6 than that of WT or inactive SGK1 (S422A; Figure 4G). Moreover, SGK1-S422D was still able to increase the ubiquitination level of SIRT6 under physiological conditions in HEK-293T cells that did not overexpress HA-Ub (Figure S2A). Even after treatment with the proteasome inhibitor MG132, SGK1-S422D was able to increase the ubiquitination of SIRT6 (Figure 4H). To determine whether SGK1-mediated phosphorylation of SIRT6 indeed promotes its degradation, WT SIRT6, SIRT6-S338A (a nonphosphorylatable mutant), and SGK1-S422D were cotransfected into HEK-293T cells. The ubiquitination of the SIRT6-S338A mutant was reduced compared with that of WT SIRT6 (Figure 4I). Furthermore, unlike the WT SIRT6 protein, SIRT6-S338A was resistant to SGK1-mediated protein degradation (Figure 4J). To determine whether SGK1-mediated SIRT6 suppression was caused by changes in protein stability, we measured the half-life of a FLAG-tagged SIRT6 in HEK-293T cells that overexpressed GFP-tagged SGK1-S422D. The half-life of SIRT6 was shorter in the presence of active SGK1 than it was in the presence of the vector (Figure 4K). However, when we overexpressed FLAG-tagged SIRT6-S338A, active SGK1 did not affect its half-life (Figure 4L). Together, these results suggest that WT SIRT6 but not SIRT6-S338A protein abundance is inhibited by SGK1 activation. Previous studies have indicated that the phosphorylation of SIRT6 at Ser338 promotes its degradation via the E3 ligase MDM2.³⁷ Therefore, we validated the role of MDM2 in this study. Following MDM2 silencing in mVSMCs, Sirt6 expression significantly increased (Figure 4M). To determine whether this effect is dependent on SIRT6 phosphorylation at Ser338, we cotransfected WT SIRT6 or the SIRT6-S338A mutant with MDM2 into HEK-293T cells. Ubiquitination assays demonstrated a reduced ubiquitination level of the SIRT6-S338A mutant (Figure 4N). Furthermore, overexpression of MDM2 resulted in a decrease in WT SIRT6 protein levels, whereas SIRT6-S338A exhibited resistance to MDM2-mediated protein degradation (Figure 4O). In summary, these data suggest that SGK1-mediated phosphorylation of SIRT6 at Ser338 increases its ubiquitination and subsequent protein degradation.

SGK1 and SIRT6 Regulate Phenotypic Transformation and Senescence in VSMCs

We further investigated the roles of SIRT6 in the phenotypic transformation of VSMCs. RT-qPCR assay found that silencing Sirt6 in mVSMCs resulted in decreased mRNA expression of the procontractile markers Myh11, Tagln, Acta2, and Cnn1 (Figure 5A). Western blot analysis showed that the protein expression levels of the contractile markers Myh11, α-Sma, and Eln decreased, while the expression levels of the synthetic markers Mgp and Opn increased after Sirt6 knockdown (Figure 5B). In addition, overexpression of SIRT6 in human aortic SMCs resulted in increased expression levels of contractile markers and decreased expression levels of synthetic markers (Figure S3A). Furthermore, immunofluorescence was used to detect changes in the fluorescence intensity of the contractile markers α-Sma and Tagln in mVSMCs. The results showed a decrease in the fluorescence intensity of α-Sma and Tagln after Sirt6 knockdown (Figure 5C and 5D). Next, EdU staining revealed Sirt6 knockdown resulted in a much higher percentage of EdU-labeled cells (Figure 5E). These findings indicate that Sirt6 deficiency promotes mVSMC proliferation. We next investigated the role of SIRT6 in VSMC aging using SA-β-gal staining. The results revealed an increase in cellular senescence after Sirt6 knockdown (Figure 5F). Western blot analysis showed that the expression of senescence markers p53, p21, and p16 increased after Sirt6 knockdown (Figure 5G). In summary, these findings suggested that SIRT6 deficiency promotes the transformation of VSMCs from a contractile to a synthetic phenotype and promotes cell proliferation and senescence.

Figure 5.

SGK1 (serum- and glucocorticoid-regulated kinase 1) and SIRT6 (sirtuin 6) regulate proliferation, senescence, and contractile-to-synthetic phenotypic transformation in mVSMCs. A through G, Control or Sirt6 small interfering RNA (siRNA)–transfected vascular smooth muscle cells (VSMCs) after 48 hours. A, Real-time quantitative polymerase chain reaction (RT-qPCR) data showing the relative mRNA expression levels of the indicated genes in Sirt6 knockdown mVSMCs. The mRNA levels were normalized to those of Gapdh. B, Western blot analysis of the indicated proteins in Sirt6 knockdown mVSMCs. β-Actin served as a loading control for Western blotting. C and D, Representative immunofluorescence staining for the contractile markers α-Sma (C) and Tagln (D) in Sirt6 knockdown mVSMCs. Scale bar=50 µm. E, Sirt6 knockdown mVSMCs were incubated with 5-ethynyl-2′-deoxyuridine (EdU) for 3 hours. A fluorescence microscope was used to detect EdU (left), and the results were statistically analyzed (right). Scale bar=100 µm. F, Representative images of SA-β-gal (senescence-associated β-galactosidase)–stained Sirt6 knockdown mVSMCs (left) and statistical analysis (right). The green regions are positively stained. Scale bar=200 µm. G, Western blot analysis of senescence markers in Sirt6 knockdown mVSMCs. β-Actin served as a loading control for Western blotting. H, Western blot analysis of the indicated proteins in mVSMCs that knock down Sgk1 or co-knock down Sgk1 and Sirt6. β-Actin served as a loading control for Western blotting. I, Representative immunofluorescence staining for the contractile marker Tagln in mVSMCs that knock down Sgk1 or co-knock down Sgk1 and Sirt6. Scale bar=25 µm. J, mVSMCs that knock down Sgk1 or co-knock down Sgk1 and Sirt6 were incubated with EdU for 3 hours. A fluorescence microscope was used to detect EdU (left), and the results were statistically analyzed (right). Scale bar=100 µm. K, Representative images of SA-β-gal stained in mVSMCs that knock down Sgk1 or co-knock down Sgk1 and Sirt6 and statistical analysis. The green regions are positively stained. Scale bar=200 µm. Data were presented as mean±SD of 3 independent experiments. A, E, and F, Statistical analyses were performed via 2-tailed unpaired t tests. J and K, Data were statistically analyzed by 1-way ANOVA followed by Tukey post hoc test. α-Sma indicates alpha smooth muscle actin; and mVSMC, primary mouse smooth muscle cells.

To further explore whether SGK1 regulates VSMC function through SIRT6, we individually knock down Sgk1, as well as simultaneously knock down Sgk1 and Sirt6 in mVSMCs. The Western blot results indicated that knockdown of Sgk1 resulted in increased expression of contractile markers Myh11, α-Sma, and Eln and decreased expression of synthetic markers Mgp and Opn. Co-knockdown of Sgk1 and Sirt6 rescued the expression of these markers (Figure 5H). Additionally, we assessed the role of a nonphosphorylated SIRT6 mutant (SIRT6-S338A) in this context by overexpressing SGK1 alone and alongside SIRT6-S338A in human aortic SMCs. The results indicated that overexpression of SGK1 decreased the expression of contraction markers Myh11, α-Sma, and Eln, while the expression of synthetic markers Mgp and Opn increased. Additionally, co-overexpression of SGK1 and SIRT6-S338A rescued the alterations in these phenotypic transformation markers induced by SGK1 overexpression (Figure S3B). These results suggest that SGK1 modulates VSMC phenotype transformation via SIRT6, and SIRT6-S338A, which cannot be phosphorylated by SGK1, can counteract the effect of SGK1 on VSMC phenotype. Immunofluorescence analysis of the contractile marker Tagln in mVSMCs showed increased fluorescence intensity upon Sgk1 knockdown, which was basically restored to control levels when Sgk1 and Sirt6 were simultaneously knocked down (Figure 5I). EdU experiments revealed that Sgk1 knockdown attenuated mVSMC proliferation, whereas simultaneous knockdown of Sgk1 and Sirt6 restored proliferation (Figure 5J). SA-β-gal staining indicated that Sgk1 knockdown slowed mVSMC senescence. Simultaneous knockdown of Sgk1 and Sirt6 restored cell senescence to levels close to normal (Figure 5K). All these experiments further suggest that SGK1 regulates the contractile-synthetic phenotypic transformation in VSMCs through SIRT6.

SIRT6 Regulates the ECM Signaling Pathway

Given the crucial roles of SGK1 and SIRT6 in VSMC phenotypic transformation, we explored the potential molecular mechanisms involved. We used high-throughput RNA sequencing to study the transcriptomic effects of knocking down SIRT6. In these experiments, siRNA was used to knock down Sirt6 expression in VSMCs. RNA extraction, purification, reverse transcription, and amplification were performed, followed by circularization and sequencing of the DNA products. Three independent samples and controls were used in these experiments. A total of 747 upregulated genes and 808 downregulated genes were identified in the Sirt6-knockdown mVSMC samples compared with the control samples (fold change, ≥1.5; Q<0.05; Figure 6A). Pathway enrichment analysis using the Kyoto Encyclopedia of Genes and Genomes database revealed that these differentially expressed genes were enriched in signaling pathways closely related to the occurrence and progression of TAD, including ECM-receptor interaction, fluid shear stress and atherosclerosis, and cellular senescence (Figure 6B). Subsequently, we selected 10 downregulated genes (Figure 6C) and 10 upregulated genes (Figure 6D) that were representative key target genes in the downstream signaling pathway regulated by SIRT6. Through literature review, genes closely related to the functions of SMCs were selected for validation, namely, Ccne2, Mef2a, Angpt2, Fgfr3, Vcl, Adcy5, Adrb2, Casp9, Cbs, and Nr4a1 and Gadd45g, Pdgfb, Pecam1, Bcar1, Ctsz, Myc, Sfn, Odc1, Cyp1a1, and Foxg1, respectively. We validated the response of these genes to Sirt6 knockdown in mVSMCs through RT-qPCR, further confirming our RNA-sequencing results. Gene set enrichment analysis also indicated that the target genes were associated with the regulation of angiogenesis, cell adhesion molecules, and the ECM signaling pathway (Figure 6E). SMCs embedded in ECM components maintain vascular homeostasis, and the phenotypic transformation of VSMCs and depletion of ECM are characteristic of aortic pathologies.^4,38 We performed heatmap analysis of the ECM pathway–related genes and the MMP family (Figure 6F and 6G). Further RT-qPCR validation after Sirt6 knockdown in mVSMCs revealed upregulation of Sdc4, Col2a1, Vwf, and Col6a5 and downregulation of Lama1, Col4a3, Fras1, Sdc1, Lama3, Sv2a, and Col4a4 (Figure 6H). Moreover, in addition to that of Mmp15, the expression of the metalloproteinase family members Mmp12, Mmp9, Mmp17, Mmp3, and Mmp8 was upregulated (Figure 6I) following Sirt6 knockdown. Additionally, knocking down Sgk1 in mVSMCs resulted in decreased expression of MMPs (Figure 6J). In summary, these findings suggest that SIRT6 inhibits MMPs, thereby participating in the regulation of the ECM signaling pathway.

Figure 6.

Whole-transcriptome identification of SIRT6 (sirtuin 6) targets. A, Heatmap of differentially expressed genes (fold change, ≥1.5; Q<0.05) in RNA-seq data from control (control-1, control-2, and control-3) and siSirt6 (siSirt6-1, siSirt6-2, and siSirt6-3) vascular smooth muscle cells (VSMCs). Blue, downregulated genes; red, upregulated genes. B, A bubble chart of the 10 enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways associated with the target genes of Sirt6. Representative genes of each pathway are also shown. The Rich factor represents the ratio of the number of target genes to the total number of genes annotated in a pathway. The Q value represents the corrected P value. C and D, Verification of the RNA-seq results via real-time quantitative polymerase chain reaction (RT-qPCR) analysis of downregulated genes (C) and upregulated genes (D). E, Gene set enrichment analysis (GSEA) of RNA-seq data. F and G, Heatmap of differentially expressed genes in the ECM (extracellular matrix) signaling pathway (F) and the MMP (matrix metalloproteinase) family (G). H and I, RT-qPCR data showing the relative mRNA expression levels of target genes in the ECM signaling pathway (H) and MMP family (I) in control or siSirt6-transfected mVSMCs. J, RT-qPCR data showing the relative mRNA expression levels of target genes of MMPs in control or siSgk1-transfected mVSMCs. C, D, and H through J, mRNA levels were normalized to those of Gapdh. Data were presented as the mean±SD of 3 independent experiments. Statistical analyses were performed via 2-tailed unpaired t tests. mVSMC indicates primary mouse smooth muscle cells; siSgk1, small interfering RNA targeting Sgk1; and siSirt6, small interfering RNA targeting Sirt6.

SGK1-SIRT6-MMP9 Axis Regulates the Phenotypic Transformation of VSMCs

Next, to identify downstream target genes directly transcriptionally regulated by SIRT6, we conducted qChIP experiments. The target genes upregulated after Sirt6 knockdown were selected, and qChIP analysis revealed Sirt6 enrichment at the promoters of Pdgfb, Myc, Sfn, Odc1, and Cyp1a1, genes associated with SMC function (Figure 7A). qChIP analysis of the SIRT6-regulated MMP gene family revealed significant enrichment of Sirt6 in the promoter regions of Mmp9 and Mmp12 (Figure 7B). Given the importance of MMP9 in VSMC phenotypic transformation, we selected MMP9 as the key target gene. Additionally, qChIP analysis demonstrated that Sirt6 binds to the Mmp9 promoter and that this is reduced upon silencing of Sirt6 expression (Figure 7C). Notably, Sirt6 depletion resulted in a significant increase in H3K27ac (acetylation of histone 3 lysine 27) at the Mmp9 promoter, while H3K9ac (acetylation of histone 3 lysine 9) and H3K18ac (acetylation of histone 3 lysine 18) showed no apparent changes (Figure 7D through 7F). In addition, the absence of Sgk1 leads to a significant increase in the enrichment of Sirt6 at the Mmp9 promoter, while the enrichment of H3K27ac at the Mmp9 promoter decreases (Figure 7G and 7H). Western blot analysis revealed that knocking down Sgk1 led to a decrease in the protein expression level of Mmp9, while knocking down Sirt6 resulted in an increase in Mmp9 protein expression. However, co-knockdown of Sgk1 and Sirt6 rescued the protein expression level of Mmp9 (Figure 7I). These findings suggested that SIRT6 directly binds to the promoter region of MMP9 by deacetylating H3K27 (histone 3 lysine 27), which results in transcriptional repression. Furthermore, the absence of SGK1 increases the enrichment of SIRT6 on the MMP9 promoter, suppressing MMP9 expression, thereby forming the regulatory axis of SGK1-SIRT6-MMP9.

Figure 7.

The Sgk1 (serum- and glucocorticoid-regulated kinase 1)-Sirt6 (sirtuin 6)-Mmp9 (matrix metalloproteinase 9) axis regulates the phenotypic transformation of mVSMCs. A, Quantitative chromatin immunoprecipitation (qChIP) analysis of indicated genes using antibodies against SIRT6 in mVSMCs. The results are presented as the fold change relative to IgG, with Gapdh serving as a negative control. B, qChIP analysis of MMPs (matrix metalloproteinases) using antibodies against SIRT6 in mVSMCs. The results are presented as the fold change relative to that of IgG, with Gapdh serving as a negative control. C through F, qChIP analysis of the recruitment of Sirt6 (C), H3K27ac (D), H3K9ac (E), and H3K18ac (F) to Mmp9 promoters in mVSMCs after transfection with control or siSirt6. The results are presented as a percentage of the input, with Gapdh serving as a negative control. G and H, qChIP analysis of the recruitment of Sirt6 (G) and H3K27ac (H) to Mmp9 promoters in mVSMCs after transfection with control or siSgk1. The results are presented as a percentage of the input, with Gapdh serving as a negative control. I, Western blot analysis of the indicated proteins in Sgk1- or Sirt6-knockdown mVSMCs. β-Actin served as a loading control for Western blotting. J, Transwell migration assays of mVSMCs following transfection with the corresponding small interfering RNA (siRNA). Migrated cells were stained and counted. The images in each group are representative of 1 field of view under the microscope. Scale bar=500 µm. K, Representative immunofluorescence staining for the contractile marker α-Sma in mVSMCs following transfection with the corresponding siRNA. Scale bar=25 µm. Data were presented as mean±SD of 3 independent experiments. A through H, Statistical analyses were performed via 2-tailed unpaired t tests. J, Data were statistically analyzed by 2-way ANOVA followed by the Tukey post hoc test. α-Sma indicates alpha smooth muscle actin; H3K9ac, acetylation of histone 3 lysine 9; H3K18ac, acetylation of histone 3 lysine 18; H3K27ac, acetylation of histone 3 lysine 27; Mvsmc, primary mouse smooth muscle cells; siSgk1, small interfering RNA targeting Sgk1; and siSirt6, small interfering RNA targeting Sirt6.

Subsequently, we assessed the impact of SGK1-SIRT6-MMP9 on the migration potential of VSMCs using transwell migration assays. The results indicated that Sgk1 knockdown reduced the migration potential of the cells, while Sirt6 knockdown increased this potential. Co-knockdown of Sgk1 and Sirt6 or of Sirt6 and Mmp9 partially rescued cell migration ability of the cells (Figure 7J), suggesting that SGK1 deficiency inhibits cell migration and SIRT6 deficiency promotes cell migration, with the SGK1-SIRT6-MMP9 axis regulating VSMC migration. Similarly, immunofluorescence experiments to detect changes in the fluorescence intensity of the contraction marker α-Sma in VSMCs revealed a notable increase in α-Sma fluorescence intensity after Sgk1 knockdown, a decrease in fluorescence intensity after Sirt6 knockdown, and partial rescue of α-Sma fluorescence intensity after simultaneous knockdown of Sgk1 and Sirt6 or Sirt6 and Mmp9 (Figure 7K). These results indicate that the SGK1-SIRT6-MMP9 axis is involved in regulating the phenotypic transformation of VSMCs.

Increased Expression of SGK1 and Decreased Expression of SIRT6 in Patients and Mice With TAD

To investigate the potential roles of SGK1 and SIRT6 in sporadic TAD, we collected control thoracic aorta and TAD tissues to evaluate the expression of SGK1 and SIRT6. Hematoxylin and eosin staining and elastic van Gieson staining revealed that in the control samples, the elastic lamina exhibited a strong and uniform structure, while in the sporadic TAD patient samples, the elastic lamina exhibited an irregular structure, increased fragmentation, and a disordered cell arrangement (characteristic of aortic dissection; Figure 8A). Further immunohistochemical staining (Figure 8B) and Western blot analysis (Figure 8C) showed that the expression of SGK1, MMP9, and MMP2 in TAD patient tissues was significantly greater than that in normal tissues, while the expression of SIRT6 was decreased. RT-qPCR was used to verify the transcription levels of the genes in aortic tissue samples from model mice. The Sgk1^F/F mice had increased Sgk1 mRNA levels after BAPN induction, while there was no significant difference in Sirt6 expression among the various groups (Figure 8D). In addition, after SMC-specific Sgk1 gene knockout in both the control and BAPN groups, the transcription level of Mmp9 decreased compared with that in Sgk1^F/F mice (Figure 8D). Furthermore, Western blot analysis revealed that, compared with that in Sgk1^F/F mice, Sirt6 expression in SMC-specific Sgk1 knockout mice was increased, Mmp9 and Mmp2 expression was decreased, and α-Sma expression was increased (Figure 8E). In summary, these findings suggest that the SGK1-SIRT6-MMP9 axis plays an important role in the development of TAD.

Figure 8.

SGK1 (serum- and glucocorticoid-regulated kinase 1) regulates SIRT6 (sirtuin 6)-MMP9 (matrix metalloproteinase 9) in patients and mice with thoracic aortic dissection. A, Representative images of hematoxylin and eosin (HE) and elastic van Gieson (EVG) staining of human thoracic aortic dissection (TAD) and control samples. Scale bar=100 µm. B, Representative images of immunohistochemical staining of SGK1, SIRT6, MMP9, and MMP2 (matrix metalloproteinase 2) in human TAD and control samples. Scale bar=100 µm. C, Western blots of SGK1, SIRT6, MMP9, and MMP2 in human TAD and control samples. β-Actin served as a loading control for Western blotting. D, Real-time quantitative polymerase chain reaction (RT-qPCR) data showing the relative mRNA expression levels of Sgk1, Sirt6, and Mmp9 in the aortas of Sgk1^F/F (Sgk1 floxed) and Sgk1^F/F;Tagln^Cre (smooth muscle cell–specific Sgk1 knockout) mice treated with control or β-aminopropionitrile monofumarate (BAPN). The mRNA levels were normalized to those of Gapdh. E, Western blots of Sgk1, Sirt6, Mmp9, Mmp2, and α-Sma in the aortas of Sgk1^F/F and Sgk1^F/F;Tagln^Cre mice treated with control or BAPN. Gapdh served as a loading control for Western blotting. F, Schematic diagram of the proposed role of SGK and SIRT6 in TAD. The proposed regulatory mechanisms of the SGK1-SIRT6-MMP9 axis in controlling vascular smooth muscle cell (VSMC) phenotypic transformation and ECM (extracellular matrix) degradation in TAD. Data were presented as mean±SD of 3 independent experiments. Statistical analyses were performed via 2-way ANOVA followed by the Tukey post hoc test. α-Sma indicates alpha smooth muscle actin.

Discussion

Currently, the understanding of the pathogenesis of TAD is limited, with previous research primarily focused on genetic risk factors. In this study, we confirmed through BAPN-induced TAD mouse model that SGK1 deficiency or inhibition reduces the risk of TAD. The high expression of SGK1 in human TAD samples demonstrated that SGK1 may be a risk factor for TAD, consistent with our findings in the mouse model, which supports the therapeutic value of targeting SGK1 for TAD treatment. Mechanistically, SGK1 promotes the ubiquitination and degradation of SIRT6 by phosphorylating Ser338 of SIRT6, thereby reducing SIRT6 protein expression. Furthermore, through epigenetic modification, SIRT6 transcriptionally suppresses the expression of MMP9, forming an SGK1-SIRT6-MMP9 regulatory axis. In addition, our data showed that SGK1 deficiency inhibits ECM degradation and VSMC phenotype transformation, which is partially dependent on the regulatory role of SIRT6-MMP9. Our proposed mechanism of action of SGK1 in TAD is shown in Figure 8F. In summary, our study not only describes the key role of SGK1 in the pathogenesis of TAD but also uncovers the potential of targeting SGK1 for TAD prevention.

Aortic aneurysm is a localized dilation and structural degeneration of specific regions of the aorta, which can occur at any part of the aorta. Blood pressure and inflammation are key factors in the progression of aneurysms to dissection.³⁹ During the onset of aortic dissection, patients exhibit altered coagulation function, with upregulation of thrombin expression.^40,41 The expression of SGK1 can be stimulated by various hormones and further enhanced by IL-6, fibroblast growth factor, platelet-derived growth factor, and thrombin.^42,43 In this study, we found that the expression of SGK1 is upregulated in the aortic walls of patients and mouse models with TAD, likely due to increased thrombin expression and elevated inflammation levels causing cellular stress during the development of TAD. Additionally, during the progression of aortic dissection, the mTOR signaling pathway is activated,⁴⁴ which subsequently activates SGK1.⁴⁵ It has been reported that upregulation of SGK1 can lead to hypertension, excessive coagulation, and fibrosis.^17,46 Therefore, we hypothesize that multiple factors lead to the increased expression of SGK1 during the onset of dissection. This upregulation of SGK1 may inhibit downstream signaling pathways and contribute to excessive coagulation and fibrosis, exacerbating the progression of aortic dissection. This regulatory process forms a loop that leads to the deterioration of TAD. Additionally, reports indicate that SGK1 promotes the proliferation of SMCs induced by low osmotic stimulation by activating the CREB (cAMP response element-binding protein) signaling pathway.⁴⁷ SGK1 promotes VSMC migration during neointima formation after arterial injury.²² Consistent with these findings, we found that SGK1 deficiency inhibited VSMC phenotypic transformation, proliferation, and migration.

Aberrant posttranslational regulation of proteins is a common feature of cardiovascular disease, and protein phosphorylation is the most prevalent, fundamental, and crucial regulatory mechanism in biological processes.⁴⁸ Our research demonstrated that SGK1 phosphorylates and inhibits the expression of SIRT6, which is an important mechanism through which SGK1 is involved in TAD occurrence and rupture. In breast cancer, MDM2 acts as an E3 ubiquitin ligase that mediates the degradation of SIRT6.³⁷ This is also confirmed in our study. As a regulator of aging, SIRT6 deficiency has been shown to increase IL-1β expression through epigenetic mechanisms in the early stages of TAD, promoting vascular inflammation, senescence, and TAD occurrence.²⁹ Additionally, previous reports have revealed other regulatory functions of SIRT6 in inflammation and senescence.^28,49 Our study shows SIRT6 plays a dual role in VSMC behavior, enhancing proliferation in early passages but accelerating senescence in later passages. Clinically, an increase in SMCs has been noted in early lesions of sporadic TAD,^46,50–52 possibly as an adaptive response to elevated stress from dynamic blood flow in dilated aortas.⁵³ However, this proliferation without functional maintenance could potentially exacerbate medial degeneration and TAD progression.⁵³ End-stage TADs exhibit significant SMC death and senescence, correlating with medial thinning and wall rupture.^29,54 Our findings align with these observations, highlighting that SIRT6 silencing not only promotes early proliferation but also accelerates senescence in later stages, reflecting the complex progression of TAD.

In this study, we found that SGK1 can inhibit the senescence phenotype of VSMCs; however, the molecular mechanisms of SGK1’s role in VSMC senescence are not well understood. Previous studies have found that SGK1 is involved in regulating oxidative stress, apoptosis, and DNA damage, all of which contribute to an accelerated senescence.⁵⁵ The SGK1-specific inhibitor EMD 638683 can clear senescent cells and improve HFD (high-fat diet)-induced pulmonary fibrosis.⁵⁶ P16 binds to the N terminus of SGK1, interfering with the interaction between E3 ubiquitin ligase NEDD4L (neural precursor cell expressed, developmentally down-regulated 4-like) and SGK1, thereby inhibiting its ubiquitin-mediated degradation, which accelerates the senescence process.⁵⁶ SGK1 is functionally and structurally similar to AKT, and they both regulate a range of fundamental cellular processes, playing a significant role in disease development.⁵⁷ In human hepatocellular carcinoma, activation of the AKT/p53/p21 signaling pathway leads to cellular senescence.⁵⁸ In psoriatic keratinocytes, AKT regulates p53/p21 expression to mediate cell senescence.⁵⁹ We hypothesize that SGK1 may promote SMC senescence by affecting signaling pathways such as p53/p21 or p16INK4a. However, the molecular mechanisms by which SGK1 affects SMC senescence require further in-depth investigation.

A key indicator of the progression of TAD is the rupture of the medial elastic layer, which is associated with elevated levels of MMPs, particularly MMP2 and MMP9.⁶⁰ The degradation of arterial elastic protein components in the ECM can promote aortic instability and plays a crucial role in various vascular diseases.⁶¹ In addition to the critical role of the ECM in maintaining vascular function, VSMCs also play an indispensable role in maintaining vascular homeostasis. Contractile VSMCs have a greater degree of differentiation and weaker proliferative and migratory abilities, while synthetic VSMCs have a lower degree of differentiation and stronger proliferative and migratory abilities; VSMC phenotypic transformation is considered to be the fundamental cause of vascular lesions.³⁴ The initial event leading to the formation of aortic dissection is the phenotypic transformation of VSMCs.⁶² This transformation of VSMCs promotes the production of proteases, which degrade the ECM, thereby weakening the aortic wall and leading to TAD rupture.⁶³ Our results showed that BAPN-treated mice exhibited significant elastin disarray and degradation, while VSMCs lacking Sgk1 exhibited a significant reversal of this change. Analysis of SIRT6 knockdown via RNA-sequencing also revealed that SIRT6 inhibits the expression of MMP family members, particularly MMP9, and suppresses the degradation of ECM components, such as Lama1 (laminin alpha-1), Col4a3 (collagen type IV alpha 3 chain), Fras1 (fraser extracellular matrix complex subunit 1), and Sdc1 (syndecan-1). Our study elucidated the mechanism of action of the SGK1-SIRT6-MMP9 transcriptional regulatory axis, which regulates the phenotypic transformation of VSMCs, participates in the ECM signaling pathway, regulates elastic layer rupture, and is involved in the TAD process.

In this study, we used the Tagln-Cre driver to achieve SMC-specific deletion of SGK1. While this approach provides valuable insights into the role of SGK1 in SMCs, we acknowledge some inherent limitations of this model. Previous studies have shown that the Tagln promoter is not exclusively targeted to SMCs; it can also be expressed in other cell types.⁶⁴ This non-SMC Tagln expression may introduce confounding factors that potentially influence the vascular wall dynamics and cell interactions before TAD development. To address these limitations, future research could use other models or those with a more precise SMC expression profile. In addition, the inhibition of SGK1 may lead to off-target vascular effects. The therapeutic application of SGK1 inhibition requires a careful evaluation of its benefits and risks. Future research needs to explore the detailed mechanisms by which SGK1 affects both vascular and nonvascular biology to better predict and manage potential off-target effects.

Our research confirmed the impact of SGK1 on the development of TAD. SGK1 promotes VSMC phenotypic transformation and ECM degradation through regulation of the SIRT6-MMP9 axis. SGK1 deficiency or pharmacological blockade inhibits vascular degeneration and TAD progression. Thus, deletion or inhibition of SGK1 may be a promising therapeutic approach for the prevention and treatment of TAD.

Article Information

Acknowledgments

The authors thank Ying Li, from the Research Center of Translational Medicine, Central Hospital Affiliated to Shandong First Medical University, for her generous assistance in the laboratory. S. Leng, W. Yu, and N. Li designed the studies and wrote the article. S. Leng, Z. Dang, and F. Zhang performed and analyzed the experiments. S. Leng, B. Shao, H. Li, and S. Xue prepared the figures. Y. Ning, X. Teng, L. Zhang, H. Wang, and P. Zhang provided reagents, advised on experimental design, and performed critical reading of the article. All authors provided critical contributions to the manuscript and approved the submitted version of the manuscript.

Sources of Funding

This work was supported by grants from the China Postdoctoral Science Foundation (2022M721334 and 2021M691226), the National Natural Science Foundation of China (82203458), the Shandong Province Postdoctoral Innovation Talent Support Plan (SDCX-ZG-202203007 and 202102045), the Science and Technology Support Plan for Youth Innovation of Colleges and Universities of Shandong Province of China (2023KJ187), the Shandong Provincial Natural Science Foundation (ZR2022QH088 and ZR2021MH385), and the Jinan Postdoctoral Innovation Project.

Disclosures

None.

Supplemental Material

Tables S1–S5

Figures S1–S3

Major Resources Table

Full Unedited Western Blot

Supplementary Material

atv-45-238-s001.pdf ^{(5.5MB, pdf)}