Exercise Attenuates Aortic Dissection Via PDE5A-Mediated Inhibition of Vascular Smooth Muscle Cell Phenotypic Switch

Abstract

BACKGROUND:

Aortic dissection (AD) is a life-threatening condition and involves pathological vascular smooth muscle cell (VSMC) phenotypic switching from a contractile to a synthetic state. Although exercise confers broad cardiovascular benefits, its role in AD pathogenesis remains unclear. This study aimed to determine whether exercise attenuates AD by modulating VSMC phenotype and to elucidate the underlying molecular mechanism.

METHODS:

Human aortic tissues from AD patients were analyzed for VSMC phenotypic markers and PDE5A (phosphodiesterase 5A) expression. A β-aminopropionitrile induced AD model was established in wild-type mice with or without treadmill exercise intervention. RNA sequencing, gain- and loss-of-function experiments, and mechanistic assays were employed to investigate the roles of PDE5A and its transcriptional regulator RUNX1 (runt-related transcription factor 1).

RESULTS:

In human AD lesional tissues, contractile VSMC markers (MYH11 [myosin heavy chain 11], CNN1 [calponin 1], and α-SMA [alpha-smooth muscle actin]) were significantly downregulated, whereas the synthetic marker osteopontin was upregulated. In β-aminopropionitrile–induced AD mice, exercise improved survival, reduced aortic dilation and AD incidence, and preserved the contractile VSMC phenotype. RNA-seq analysis identified PDE5A as a key exercise-responsive gene. PDE5A expression was reduced in human AD lesional tissues and β-aminopropionitrile–treated mice but was upregulated by exercise. VSMC-specific overexpression of PDE5A attenuated AD progression, whereas PDE5A inhibition abolished the attenuating effects of exercise. We further identified RUNX1 as a transcriptional repressor of PDE5A that is upregulated in AD conditions and suppressed by exercise. Inhibition of RUNX1 upregulated PDE5A expression, preserved VSMC contractility, and reduced AD incidence.

CONCLUSIONS:

In conclusion, we identify a novel RUNX1-PDE5A axis that mediates the beneficial effects of exercise against AD. Exercise attenuates AD by reducing RUNX1-mediated transcriptional repression of PDE5A, thereby maintaining VSMC contractile phenotype. These findings highlight the RUNX1-PDE5A pathway as a promising preventive target.

What Are the Clinical Implications?

This study elucidates a previously unknown molecular mechanism through which exercise attenuates the development of aortic dissection. We demonstrate that aerobic exercise promotes aortic wall integrity by inhibiting the transcription factor RUNX1 (runt-related transcription factor 1), thereby upregulating the enzyme PDE5A (phosphodiesterase 5A) in vascular smooth muscle cells. The RUNX1-PDE5A axis is essential for maintaining the contractile vascular smooth muscle cell phenotype. Our findings provide a mechanistic rationale for implementing structured, moderate-intensity exercise as a preventive strategy in high-risk individuals. Furthermore, by defining this pathway, our work identifies RUNX1 and PDE5A as promising therapeutic targets. Pharmacological modulation of the RUNX1-PDE5A axis could lead to novel drug-based strategies for patients unable to engage in rigorous physical activity, offering a potential alternative to stabilize the aortic wall.

See accompanying editorial by Prakash

Aortic dissection (AD) represents a catastrophic cardiovascular emergency marked by disruption of the medial layer and progressive dilation of the aortic wall. Without prompt intervention, early mortality in AD increases by ≈1% to 2% per hour following symptom onset.¹ Vascular smooth muscle cells (VSMCs), which reside predominantly within the medial layer of blood vessels, are central not only to the regulation of vascular tone and blood pressure but also to the pathogenesis of aortic diseases, including AD. The development of AD involves progressive impairment of VSMC contractile function and degradation of the extracellular matrix (ECM), driven largely by a pathological phenotypic switch of VSMCs: from a quiescent, contractile state to a proliferative, synthetic phenotype.^2–4 Accumulating evidence indicates that inhibition of VSMC phenotypic switching can attenuate both the incidence and progression of AD.^5,6 While surgical intervention remains the mainstay of clinical management for AD, effective pharmacotherapies for prevention or treatment are currently lacking. Thus, a deeper understanding of the molecular mechanisms governing VSMC phenotypic transition is essential for developing novel preventive and therapeutic strategies against AD.

Physical activity is widely recognized as a cornerstone of cardiovascular health, with well-established benefits in the management of hypertension, hyperlipidemia, heart failure, and vascular aging.^7–10 Defined as any bodily movement that increases energy expenditure above basal metabolic levels, physical activity encompasses a spectrum of daily activities.^8,9 In increasingly sedentary populations, structured exercise represents a key strategy for achieving beneficial levels of physical activity. Experimental studies have demonstrated that moderate aerobic exercise attenuates aortic root dilation, reduces the incidence of AD and aneurysm, and mitigates ECM degradation in relevant preclinical models.^11,12 Although early postoperative exercise training has been shown to be safe, feasible, and beneficial for functional recovery in patients after AD repair,^13,14 the role of exercise in the primary pathogenesis and progression of AD remains poorly defined. Emerging evidence suggests that exercise helps preserve VSMC contractility and attenuate maladaptive vascular remodeling in hypertension, partly through Akt-dependent signaling.^15,16 However, whether exercise modulates AD development, particularly through regulation of phenotypic switching, remains unexplored.

PDE5A (phosphodiesterase 5A) is a cyclic guanosine monophosphate (cGMP)-specific phosphodiesterase that plays a crucial role in cellular signal transduction by hydrolyzing cGMP and thereby regulating its intracellular concentrations.^17,18 Interestingly, the PDE5A inhibitor sildenafil, originally developed for angina, was serendipitously found to enhance penile erection via cGMP-mediated vasodilation, leading to its repurposing for erectile dysfunction and subsequent approval for pulmonary arterial hypertension.¹⁸ By inhibiting cGMP degradation, PDE5A inhibitors amplify nitric oxide signaling and induce vasodilation in VSMCs. Emerging evidence underscores the significance of PDE5A in cardiovascular pathophysiology.¹⁷ For instance, in heart failure, PDE5A upregulation may impair cGMP-PKG (protein kinase G) signaling and exacerbate cardiac dysfunction, whereas its inhibition confers protective effects in preclinical models.^17,19 In the pulmonary circulation, PDE5A inhibition reduces pulmonary vascular resistance and improves hemodynamics in pulmonary hypertension.¹⁸ PDE5A is highly expressed in VSMCs and contributes to vascular relaxation, aortic development, and ECM homeostasis.^20,21 Recent studies further associate reduced PDE5A activity with vascular pathologies, including aortic aneurysm and dissection,^20,21 but its role in AD and potential modulation through exercise remain unknown.

Transcriptional regulation of PDE5A in VSMCs remains incompletely characterized. RUNX1 (Runt-related transcription factor 1), a master regulator of hematopoiesis and leukemogenesis, orchestrates diverse cellular processes through context-specific transcriptional programs.^22,23 This transcription factor comprises 3 major isoforms (RUNX1a/b/c) generated via alternative promoter usage and splicing: RUNX1a and RUNX1b are transcribed from the P2 promoter, whereas RUNX1c, derived from the P1 promoter, represents the most abundant isoform in definitive hematopoiesis.^24–26 Although extensively studied in hematologic malignancies, emerging evidence suggests a role for RUNX1 in vascular pathophysiology.^27,28 Recent studies show that endothelial RUNX1 modulates nitric oxide signaling through m6A-dependent mRNA stabilization and participates in blood pressure regulation.²⁷ Beyond the endothelium, however, the functions of RUNX1 in VSMCs remain poorly defined, though its dysregulation has been implicated in aortic aneurysm development.²⁸ Whether RUNX1 contributes to AD pathogenesis through direct actions in VSMCs, and the underlying mechanisms involved, have not yet been investigated.

In this study, we aimed to test the hypothesis that exercise attenuates AD by preserving the VSMC contractile phenotype. We further sought to investigate whether PDE5A and its upstream transcriptional regulators serve as the key mechanistic mediators of this protection.

Methods

Data Availability

The authors declare that all supporting data are available within the article (and its Supplemental Material). The raw RNA-sequencing data have been deposited in the Genome Sequence Archive²⁹ in the National Genomics Data Center,³⁰ China National Center for Bioinformation/Beijing Institute of Genomics, Chinese Academy of Sciences (GSA: CRA097761), which are publicly accessible at https://ngdc.cncb.ac.cn/gsa. The Major Resources Table, which provides detailed information on the resources used in this study, can be found in the Supplemental Material.

Human Subjects

All human samples were collected from patients presented to the Department of Cardiovascular Surgery at Tianjin Medical University General Hospital (Table S1). The samples in the AD group were obtained from patients diagnosed with thoracic AD via aortic computed tomography angiography who underwent Sun’s procedure. Control specimens consisted of histologically normal aortic tissues harvested from distal anatomic sites in these same individuals. Immediately after harvest, aortic tissues were immersed in ice-cold saline, cleansed of thrombi through dissection, and subsequently fixed in 4% paraformaldehyde. Following paraffin embedding, tissues were sectioned (4 µm) continuously for pathological analysis. Alternatively, samples were flash-frozen in liquid nitrogen for subsequent protein or mRNA expression quantification. The study was conducted in accordance with the ethical guidelines of the 1975 Declaration of Helsinki, and the study protocol was approved by the Medical Ethics Committee of Tianjin Medical University General Hospital (IRB2025-YX-140-01).

Animal Studies

Previous studies have shown that sex can influence the prevalence of AD.³¹ To minimize the impact of sex hormones and ensure a high incidence of AD, only male animals were used in this study. Male C57BL/6J mice (3–4 weeks old) were purchased from Gempharmatech Co, Ltd (Nanjing, China). All mice were housed under temperature- and humidity-controlled specific pathogen-free conditions with a 12-hour light/12-hour dark cycle. All animal experiments were conducted by 2 independent investigators in a blinded fashion to minimize potential bias in data collection. Animals were randomly assigned to experimental groups based on body weight using the Excel RAND function, and group allocation was concealed from the investigator responsible for treatment administration. Researchers remained blinded to mouse treatment groups throughout the experiment and data analysis. Sample sizes per group were determined empirically, based on standard practices in the field and our long-standing experimental experience. Each animal was considered an independent unit for statistical analysis. All animal studies were conducted with the approval of the Institutional Animal Care and Use Committee of Tianjin Medical University General Hospital (IRB2025-DW-28), and all animal procedures conformed to the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

For the β-aminopropionitrile (BAPN) fumarate (A3134, Sigma-Aldrich, St Louis) induced AD model, 3 to 4-week-old male C57BL/6J mice were treated with BAPN (1 g/kg per day)-supplemented water or normal standard drinking water for 4 or 12 weeks. At 4 or 12 weeks after BAPN administration, mice were anesthetized for aortic tissue collection. AD was defined as the formation of a blood-filled false lumen within the aortic media or massive hemothorax due to aortic rupture. Necropsies were performed on all mice that died before the predetermined end point, regardless of suspected cause.

Regarding the implementation of the aerobic exercise training protocol, all mice were subjected to forced treadmill exercise or free activity for 12 weeks. For mice undergoing aerobic exercise training, a 1-week adaptation period (5 d/wk) was first implemented. This began with exposure to the treadmill, gradually accelerating to 5 m/min for 5 minutes, followed by 10 m/min for 20 minutes and 5 m/min for 5 minutes. Subsequently, a 12-week main training phase (5 d/wk) was conducted, starting again with treadmill exposure, gradually accelerating to 5 m/min for 5 minutes, then progressing to 15 m/min for 20 minutes, followed by 5 m/min for 5 minutes.

The maximal diameters of the thoracic aorta were measured as described previously.³² Measured the diameters of multiple aortic locations, including the ascending aorta, aortic arch, and descending thoracic aorta, and selected the largest diameter as the maximum diameter of the thoracic aorta.

Age- and sex-matched mice were included in all experimental groups. Exclusion criteria were limited to preexisting health conditions unrelated to the study, and no animals met these criteria.

Sildenafil Administration

Stock solution of sildenafil (HY-15025A; MedChemExpress, NJ) was prepared in dimethyl sulfoxide, stored at −80 °C, and diluted with PEG300, Tween 80, and sterile saline before intraperitoneal administration. Mice received intraperitoneal injections of sildenafil at a dose of 20 mg/kg every other day for 12 weeks.³³ Control mice were administered an equivalent volume of the vehicle solution. Mice were weighed before each injection.

Ro5-3335 Injection

Stock solution of Ro5-3335 (HY-108470, MedChemExpress, NJ) was prepared in dimethyl sulfoxide, stored at −80 °C, and diluted with PEG300, Tween 80, and sterile saline before intraperitoneal administration. Mice were injected subcutaneously with Ro5-3335 at 20 mg/kg every other day for 4 weeks.³⁴ Control animals received an equal volume of the vehicle solution. Mice were weighed before each injection.

Isolation and Culture of Primary RASMCs

Primary rat aortic smooth muscle cells (RASMCs) were isolated from the thoracic aortas of Sprague-Dawley rats by collagenase digestion as described previously.³⁵ Briefly, male Sprague-Dawley rats (weight, 150–180 g) were dissected to expose the thoracic cavity. Thoracic aortas were excised and rinsed with ice-cold sterile PBS. After gently removing the adventitia and intima, the medial layer was cut into fragments of 1 to 2 mm. Tissue fragments were digested with 0.1% collagenase type II (40508es60; Yeasen Biotech, Shanghai, China) at 37 °C with 5% CO₂ in an incubator for 2 hours. Digestion was terminated by adding an equal volume of FBS (A5256701; Gibco BRL, Grand Island, United States). Cells were pelleted by centrifugation (300×g, 5 min) and resuspended in low-glucose DMEM (11885084; Gibco BRL, Grand Island, United States) containing 20% FBS for culture incubation. RASMCs at passages 3 to 6 were used for all experiments. The purity of RASMCs was tested by immunofluorescence staining for α-SMA (α‐smooth muscle actin), and the positivity rate reached over 95%.

Induction of RASMCs Phenotypic Switch

All experiments were conducted using RASMCs between passages 3 and 6. RASMCs grew to 80% to 90% confluence and serum-starved for 24 hours, followed by PDGF-BB (platelet-derived growth factor-BB, 100-14B, PeproTech Inc, NJ; 20 ng/mL) induction for 48 hours. RASMCs administered with vehicle served as control. Following stimulation with vehicle or PDGF-BB, RASMCs were subjected to further experimentation.

Quantitative Real-Time Polymerase Chain Reaction

RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR) were performed as previously described. Briefly, total RNA was extracted using TRIzol Reagent (15596018; Invitrogen, Carlsbad, United States), and RNA concentration was measured using a NanoDrop (Thermo Fisher Scientific, Waltham, United States). A total of 1 μg of RNA was used to remove genomic DNA and was reverse transcribed into cDNA using cDNA Synthesis SuperMix (11141es60; Yeasen Biotech, Shanghai, China). SYBR Green Master Mix (11184es08; Yeasen Biotech, Shanghai, China) was applied for qRT-PCR reaction to measure the expression of genes using a LightCycler 480 (Roche, Basel, Switzerland). The mRNA expression levels of target genes were normalized to those of Gapdh. The primer sequences used in this study are listed in Table S2.

Western Blot Analysis

Protein extraction and Western blot were performed as previously described.³⁶ Briefly, total proteins were extracted from RASMCs or aortic tissues using RIPA buffer with protease inhibitors, and protein concentrations were quantified using the BCA Protein Assay Kit (23225; Thermo Fisher Scientific, Waltham, United States). Twenty micrograms of total protein sample were loaded and separated electrophoretically on SDS-polyacrylamide gel electrophoresis gels and then transferred to polyvinylidene fluoride membranes (IPVH00010, Merck Millipore, Billerica, United States). Subsequently, the membrane was blocked with 5% skimmed milk at room temperature for 2 hours and incubated at 4 °C overnight with the following primary antibodies: MYH11 (myosin heavy chain 11; catalog no: A10827; Polyclonal, ABclonal Technology, Wuhan, China), CNN1 (calponin 1; catalog no: A16638; Polyclonal, ABclonal Technology, Wuhan, China), α-SMA (alpha-smooth muscle actin; catalog no: A2547; clone no: 1A4, Sigma-Aldrich, St Louis, United States), OPN (osteopontin; catalog no: 83341-1-RR, clone no: 240206A2, Proteintech, Wuhan, China), PDE5A (catalog no: A6831, Polyclonal, ABclonal Technology, Wuhan, China), RUNX1 (catalog no: 25315-1-AP, Polyclonal, Proteintech, Wuhan, China), α-tubulin (catalog no: AC012, Clone No: AMC0479, ABclonal Technology, Wuhan, China), and GAPDH (glyceraldehyde-3-phosphate dehydrogenase; catalog no: AC002, Clone No: AMC0062R, ABclonal Technology, Wuhan, China). Membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies (catalog no: 7076, Cell Signaling Technology, Boston, United States; catalog no: 111-035-003, Jackson ImmunoResearch, Lancaster, United States) at room temperature for 2 hours, and protein bands were detected using an enhanced chemiluminescence assay (34580; Thermo Fisher Scientific, Waltham, United States). Images were obtained using a Tanon Imaging system (Tanon 5200 Multi, Shanghai, China), and the blots were quantitatively analyzed using the FIJI ImageJ software (http://imagej.net/Fiji).

Blood Pressure Measurement

Systolic blood pressure (SBP) was measured in conscious mice using a noninvasive tail-cuff system (Visitech Systems BP-2000, Apex, NC). Measurements were performed weekly between 9:00 and 11:00 am to control for diurnal variation. Mice were acclimated to short-term restraint before measurement. For each recording session, animals were placed in individual holders on a temperature-controlled platform (37 °C) to maintain steady-state conditions. Ten consecutive readings were obtained per mouse per session. The mean SBP was calculated from the last 8 stable readings, excluding outliers caused by excessive movement. The technician performing the measurements and analyzing the raw data was blinded to the experimental group allocation throughout the study.

Histological Analysis

Following euthanasia, mouse thoracic aortas were isolated and rinsed with precooled PBS. Mice thoracic aorta tissues were fixed in 4% paraformaldehyde at room temperature for at least 24 hours, and then underwent graded ethanol dehydration, paraffin embedding, followed by sectioning into 4 µm slices for subsequent histological staining. A hematoxylin and eosin staining kit (G1076; Servicebio, Wuhan, China) was used for morphometric analysis. An elastin Van Gieson staining kit (G1042; Servicebio, Wuhan, China) was used for elastin assessment. A Masson trichrome staining kit (G1006; Servicebio, Wuhan, China) was used for collagen assessment. The elastic fiber quantification is a grade for the entire section, and the elastin degradation grades are as follows: 1, no degradation; 2, mild elastin degradation; 3, severe elastin degradation; and 4, aortic rupture.³⁷

RNA Sequencing Analysis

Mice thoracic aortas were sent to Beijing Novogene Biomedical Technology Co., Ltd. for transcriptome sequencing. Briefly, Mice thoracic aortas were collected, and the adventitia was carefully removed. Following total RNA extraction, Poly(A)⁺ mRNA was purified from total RNA using oligo(dT) magnetic beads and fragmented randomly. First-strand cDNA synthesis employed reverse transcriptase, followed by second-strand synthesis with DNA Polymerase I. The resulting double-stranded cDNA underwent end repair, 3’ end adenylation, and ligation to Illumina universal adapters. After PCR amplification, libraries were purified with AMPure XP beads, quantified using an Agilent 2100 Bioanalyzer and Qubit 2.0 Fluorometer, and sequenced on an Illumina NovaSeq 6000 platform. Genes with adjusted P<0.05 and |log2 (fold change) |>0.5 were deemed to be differentially expressed. Data were analyzed using the NovoMagic cloud platform (http://magic.novogene.com/).

Immunofluorescence Staining

For immunofluorescence staining, paraffin-embedded sections (4 µm) were deparaffinized and rehydrated, followed by antigen retrieval using citrate buffer. Subsequently, sections were blocked with 3% BSA at room temperature for 1 hour. The sections were incubated with primary antibodies against PDE5A (1:400; catalog no: 85030-1-RR, clone no: 242597B10, Proteintech, Wuhan, China) and α-SMA (1:500; catalog no: A2547, Clone No: 1A4, Sigma-Aldrich, St Louis, United States) to specifically detect proteins at 4 °C overnight, and then for 1 hour with secondary antibodies while being protected from light. The sections were incubated with DAPI-containing anti-fluorescence quenching sealing tablets, observed, and photographed using a confocal microscope (Carl Zeiss, Jena, Germany).

AAV-Mediated Pde5a Overexpression In Vivo

To overexpress Pde5a, 2-week-old male C57BL/6 mice received intravenous injections of recombinant adeno-associated virus (AAV) serotype 9 (2×10¹¹ vector genomes copies per mouse) expressing mouse Pde5a under the VSMC-specific Tagln promoter. All AAV9 constructs were generated by Hanheng Biotechnology Co, Ltd (Hangzhou, China). Two-week postinjection, aortic PDE5A overexpression efficiency was validated by Western blot analysis.

Lentiviral-Mediated Pde5a Overexpression In Vitro

For lentiviral transduction, RASMCs were incubated with lentivirus containing empty vector or rat Pde5a-encoding plasmids. The multiplicity of infection (MOI), defined as the ratio of transducing lentiviral particles to the number of cells, was set at 20. At 40% confluence, RASMCs were transduced with lentivirus in 50% normal culture volume, supplemented with polybrene. After 4 hours of incubation, an equal volume of prewarmed complete medium was added. Culture medium containing lentivirus was replaced with fresh complete medium at 24 hours posttransduction. After 48 hours of transfection, cells were processed for downstream assays.

Chromatin Immunoprecipitation Coupled With qRT-PCR

RASMCs were crosslinked with 1% formaldehyde for 10 minutes at room temperature to fix DNA-protein interactions, and the reaction was quenched with 125 mmol/L glycine through competitive aldehyde neutralization. Following harvest and lysis, fixed cells underwent chromatin shearing via 30 cycles of sonication (30s on and 30s off), generating ≈300 bp chromatin fragments. For immunoprecipitation, the lysates were incubated with anti-RUNX1, or normal IgG antibodies (30000-0-AP; Proteintech, Wuhan, China) overnight at 4°C, followed by incubation with protein A/G MagBeads (36417ES03; Yeasen Biotech, Shanghai, China) for 2 hours. Immunocomplexes and input controls were eluted in TE buffer (10 mmol/L Tris-HCl, 1 mmol/L EDTA, pH 8.0) and reverse-crosslinked overnight at 55 °C in elution buffer (1% SDS, 100 mmol/L NaHCO₃). DNA was purified using a DNA purification kit (D0033; Beyotiome Biotechnology, Shanghai, China). Chromatin Immunoprecipitation coupled with qPCR analysis used SYBR Green Master Mix (11184es08; Yeasen Biotech, Shanghai, China) with primers listed in Table S3.

Statistical Analyses

Continuous variables were summarized as mean values±SEM and checked for normality using the Shapiro-Wilk test to check for conformity to the normal distribution. Comparisons between 2 groups were analyzed using Student t tests. Multiple-group analyses were compared with 2-way ANOVA with Bonferroni posthoc correction. The statistical significance level was set at 0.05 unless otherwise stated. All statistical analyses were performed, and graphs were generated with GraphPad Prism 10 (GraphPad Software Inc, CA).

Results

VSMC Phenotypic Switch Occurs in AD and Is Attenuated by Exercise in Mice

Under pathological stress, VSMCs undergo a phenotypic switch from a contractile to a synthetic phenotype, contributing to impaired vascular function.³⁸ To characterize VSMC phenotypic switching in AD, we first analyzed human aortic tissues obtained from patients with hypertension-associated thoracic AD (clinical characteristics summarized in Table S1). Using paired samples, lesional tissue from the dissection site (AD-Lesion) and histologically normal tissue from a distal segment of the same aorta (AD-Distal Non-Lesion), we performed quantitative real-time polymerase chain reaction (qRT-PCR). The result revealed significantly reduced mRNA levels of key contractile markers, including myosin heavy chain 11 (MYH11), actin alpha 2, smooth muscle (ACTA2), myosin light chain 9 (MYL9), calponin 1 (CNN1), and transgelin (TAGLN), in AD lesions compared with their paired distal controls. Conversely, the synthetic marker secreted phosphoprotein 1 (SPP1) was markedly elevated (Figure 1A). Consistent with these transcriptional changes, Western blotting confirmed substantially reduced protein expression of MYH11, CNN1, and α-SMA, along with increased OPN levels in AD-lesion tissues (Figure 1B and 1C), indicating that VSMC phenotypic switching occurs during human AD pathogenesis.

Figure 1.

Aerobic exercise preserves the contractile phenotype of vascular smooth muscle cells (VSMCs) in the mouse aorta. A, Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis of MYH11, ACTA2, MYL9, CNN1, TAGLN, and SPP1 mRNA levels in distal nonlesion tissue (AD-distal non-lesion; n=5) and lesion tissue (AD-lesion; n=5) from patients with AD. B and C, Western blot analysis (B) and quantification (C) of MYH11 (myosin heavy chain 11), CNN1 (calponin 1), α-SMA (α‐smooth muscle actin), and OPN (osteopontin) protein expression in human aortic specimens (n=8). D through G, Three-week-old mice underwent 1 week of acclimation training followed by administration of β-aminopropionitrile (BAPN; 1 g/kg per day) or normal drinking water, combined with 12 weeks of either forced treadmill exercise training (Run) or sedentary (Sed) conditions. D, Schematic of the experimental timeline. E, qRT-PCR analysis of Myh11, Myl9, Acta2, Cnn1, Tagln, and Spp1 mRNA levels in mouse aortic tissues (n=6). F and G, Western blot analysis (F) and quantification (G) of MYH11, α-SMA, CNN1, and OPN protein levels in mouse aortic tissues (n=6). Data are presented as mean±SEM. An unpaired Student t test was performed in A and C, 2-way ANOVA followed by a Tukey multiple-comparison test was used in E and G. Panel D created in BioRender. Mao, J. (2026) https://BioRender.com/qlycz8m.

To further examine this transition and evaluate the influence of exercise, we established a mouse model of AD using BAPN administered via drinking water, with control groups receiving normal drinking water; mice were assigned to either a sedentary (Sed) or treadmill exercise (Run) group (Figure 1D). BAPN+Sed mice exhibited significantly decreased expression levels of contractile markers (Myh11, Myl9, Acta2, Cnn1, Tagln) and increased expression of the synthetic marker Spp1 compared with Sed controls (Figure 1E). In contrast, BAPN+Run mice displayed significant retention of contractile marker expression and reduced Spp1 levels relative to BAPN+Sed groups (Figure 1E). These mRNA-level findings were corroborated by parallel changes at the protein level (Figure 1F and 1G). Furthermore, exercise markedly attenuated the inflammatory response in aortas from BAPN-induced AD mice (Figure S1). Collectively, these results demonstrate that AD is associated with a shift of VSMCs toward a synthetic phenotype and that exercise effectively attenuates this pathological transition.

Exercise Attenuates BAPN-Induced AD Incidence and Severity in Mice

We next evaluated the impact of exercise on AD development in a BAPN-induced mouse model. Consistent with previous reports,¹¹ BAPN treatment induced lethal AD in 13 of 44 Sed mice by day 28, with this number increasing to 23 of 44 by day 84. Furthermore, BAPN significantly increased the maximal thoracic aortic diameter and induced AD in 31 of 44 mice, with aortic rupture occurring in 23 of 44 mice. Histological analysis via hematoxylin and eosin staining revealed marked medial thickening, elastic Verhoeff-Van Gieson (elastin Van Gieson), and Masson trichrome staining showed pronounced fragmentation of elastic fibers and increased collagen deposition in BAPN+Sed mice (Figure 2A through 2H). Exercise intervention substantially improved survival: after 84 days of BAPN challenge, 23 of 44 mice in the BAPN+Sed group died, compared with 8 of 37 in the BAPN+Run group (P<0.05; Figure 2A). Notably, SBP did not differ significantly between groups, indicating that the attenuating effect of exercise was not mediated by changes in hemodynamic load (Figure 2B). Moreover, exercised mice exhibited attenuated pathological severity: compared with BAPN+Sed controls, BAPN+Run mice displayed reduced aortic dilation, as indicated by decreased maximal thoracic aortic diameter (Figure 2C and 2D), along with significantly lower AD incidence (13 of 37 versus 31 of 44; P<0.05) and rupture rate (8 of 37 versus 23 of 44; P<0.05; Figure 2E and 2F). Histological examination of the suprarenal aorta demonstrated pronounced medial disruption and elastin fragmentation in BAPN+Sed mice, both of which were markedly ameliorated by exercise (Figure 2G). Quantification of elastin degradation further confirmed this protective effect (Figure 2H). Together, these data underscore the beneficial role of exercise in limiting AD development under pathological stress.

Figure 2.

Aerobic exercise attenuates thoracic aortic dissection in β-aminopropionitrile (BAPN)–induced mice. A, Survival curves of mice receiving normal drinking water or BAPN water, combined with either forced treadmill exercise (Run) or sedentary (Sed) conditions. Survival data were analyzed by the Kaplan-Meier method and compared using log-rank tests (n=15-44). B, Systolic blood pressure at 0, 1, 2, 3, and 4 weeks after BAPN administration in mice from the indicated experimental groups (n=8). C, Representative images of aortas from the indicated experimental groups. D, Maximal thoracic aorta diameter in mice receiving normal standard drinking water or BAPN water (n=8). E, The incidence of aortic dissection formation in mice from the indicated experimental groups (n=15–44). F, The incidence of aortic rupture in mice from the indicated experimental groups (n=15–44). G and H, Representative Hematoxylin and Eosin (H&E), elastic Verhoeff-Van Gieson (EVG), and Masson trichrome staining of the thoracic aortas in mice receiving normal standard drinking water or BAPN water (G), and quantification of elastin degradation based on a 4‐point scoring system (H; n=6). Scale bar=100 µm. Data are presented as mean±SEM. Two-way ANOVA followed by Tukey multiple-comparison test was used in B, D, and H; Fisher exact test was performed in E and F.

PDE5A Is Downregulated in AD and Restored by Exercise

To elucidate the molecular mechanisms underlying exercise-induced aortic protection, we performed transcriptome sequencing (RNA-seq) on aortic tissues from mice subjected to BAPN treatment with or without exercise (Figure 3A). Distinct gene expression patterns among experimental groups were visualized using a heatmap, providing a foundation for investigating exercise-mediated aortic protection (Figure S2A). Comparative analysis revealed 3274 differentially expressed genes (DEGs) in BAPN+Sed mice compared with the Sed group (log₂ [fold change] ≥0.5, −log₁₀ [q value] ≥1.3), including 1164 downregulated genes, among them, key VSMC contractile markers such as Myh11, Myl9, Tagln, and Cnn1, and 2110 upregulated genes (Figure S2B). In contrast, comparison between the BAPN+Run and BAPN+Sed groups identified 3510 DEGs, with 1067 genes upregulated, including the same contractile markers, and 2443 downregulated (Figure 3B).

Figure 3.

PDE5A (phosphodiesterase 5A) is involved in the aortic protective effect of aerobic exercise in β-aminopropionitrile (BAPN)–induced mice. A, Schematic diagram of RNA sequencing (RNA-seq) performed on mouse aorta from mice receiving either normal drinking water or BAPN water and simultaneously undergoing either forced treadmill exercise (Run) or sedentary (Sed) conditions, (n=3). B, Volcano plot of the differentially expressed genes (DEGs) between BAPN+Run and BAPN+Sed mice. C, Venn diagram identifying overlapping genes among genes of vascular smooth muscle cell (VSMC) contractile phenotype-related (PRJNA871790), differentially expressed markers for VSMCs (GSE207784), DEGs in human aortic dissection (AD, GSE153434), and DEGs in mouse AD. D, Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis of Pde5a mRNA levels in indicated groups (n=6). E and F, Western blot analysis (E) and quantification (F) of PDE5A protein in indicated groups (n=6). G, qRT-PCR analysis of PDE5A mRNA levels in distal nonlesion tissue (AD-distal non-lesion; n=5) and lesion tissue (AD-lesion; n=5) from patients with aortic dissection. H and I, Western blot analysis (H) and quantification (I) of PDE5A protein in human aortic specimens (n=8). J and K, Representative immunofluorescence images (J) and quantification (K) of PDE5A (red), α-SMA (α‐smooth muscle actin; green), and nuclei (DAPI, blue) in distal nonlesion tissue (n=3) and lesion tissue (n=3) from patients with AD. Scale bar=100 µm. Data are presented as mean±SEM. Two-way ANOVA followed by Tukey multiple-comparison test was used in D and F, an unpaired Student t test was performed in G, I, and K. Panel A created in BioRender. Mao, J. (2026) https://BioRender.com/qlycz8m.

Gene ontology enrichment analysis of DEGs between BAPN+Run and BAPN+Sed mice revealed significant enrichment in biological processes related to cell migration, adhesion, and differentiation (Figure S2C). We further analyzed a published RNA-seq data set (PRJNA871790) to examine transcriptional changes in RASMCs induced by TGF-β (transforming growth factor-β) or PDGF-BB (Figure S2D). A subset of genes downregulated by TGF-β and upregulated by PDGF-BB was defined as synthetic phenotype-related genes (Figure S2E). Gene set enrichment analysis using this 1347-gene set demonstrated significant enrichment of synthetic phenotype-related genes both in BAPN-induced mice and in mice where exercise reversed this effect (Figure S2F), supporting a key role for exercise in modulating VSMC phenotypic switching.

To identify genes associated with contractile phenotype maintenance, we defined a gene set upregulated by TGF-β and downregulated by PDGF-BB in RASMCs as contractile phenotype-related genes (PRJNA871790; Figure S3A). We also identified: genes downregulated in BAPN+Sed versus Sed as mouse AD-DEGs (Figure S3B); genes downregulated in VSMCs from human aortic aneurysm tissues as VSMC markers (GSE207784; Figure S3C); and genes downregulated in human AD tissues as human AD-DEGs (GSE153434; Figure S3D). Integrative Venn analysis of these 4 gene sets identified 25 consensus candidates (Figure 3C). KEGG pathway analysis of upregulated genes in the BAPN+Run group highlighted enrichment of cGMP-PKG signaling and vascular smooth muscle contraction (Figure S3E). Among the top candidate genes, PDE5A was prioritized due to its established role in regulating cellular cGMP levels through phosphodiesterase activity and its close association with cGMP-PKG signaling.¹⁷ Consistently, Pde5a expression was reduced in BAPN-treated mice and elevated by exercise (Figure S2B; Figure 3B).

We next validated PDE5A expression across experimental groups. Both mRNA and protein levels of PDE5A were significantly downregulated in aortas from BAPN+Sed mice compared with Sed controls and were elevated in BAPN+Run mice (Figure 3D through 3F; Figure S3F and S3G). Importantly, the downregulation of PDE5A observed in murine AD was also recapitulated in human disease. In the same set of paired human aortic samples, PDE5A mRNA and protein levels were markedly lower in dissection lesions compared with distal nonlesioned aortic tissue (Figure 3G through 3I). Immunofluorescence staining confirmed that PDE5A co-localized with α-SMA in VSMCs and showed a clear decrease in signal intensity within the medial layer of dissected aortas (Figure 3J and 3K). This parallel downregulation in human AD tissues reinforces the translational relevance of PDE5A deficiency in the pathophysiology of AD and aligns with the concomitant shift in VSMC phenotype. Together, these findings indicate that PDE5A expression is suppressed during AD pathogenesis and is upregulated by exercise, suggesting a pivotal role for PDE5A in mediating the protective effects of exercise against AD.

PDE5A Overexpression Inhibits VSMC Phenotypic Switching in VSMCs

To determine the functional role of PDE5A in VSMC phenotypic switching, we isolated and identified RASMCs (Figure S4A and S4B) and treated them with PDGF-BB to induce a synthetic phenotype.³⁹ As anticipated, PDGF-BB treatment promoted a transition from a contractile to a synthetic state, as evidenced by downregulated expression of contractile markers and upregulated expression of synthetic markers at both mRNA and protein levels (Figure S4C through S4E). Furthermore, PDGF-BB stimulation also increased the expression of proinflammatory genes, including interleukin 6 (Il-6) and matrix metalloproteinase 9 (Mmp9), in RASMCs (Figure S4F). Concomitantly, PDGF-BB significantly reduced both mRNA and protein levels of PDE5A, recapitulating the downregulation observed in the BAPN-induced mouse AD model (Figure S4G through S4I). To assess whether PDE5A modulates this phenotypic switching process, we transduced RASMCs with a lentivirus encoding rat PDE5A, which efficiently increased PDE5A expression (Figure S5A). Notably, PDE5A overexpression attenuated the PDGF-BB-induced phenotypic transition, manifested as upregulated expression of contractile markers and downregulated expression of synthetic markers (Figure S5B through S5D), and abrogated PDGF-BB-induced cell proliferation (Figure S5E). Furthermore, PDE5A overexpression suppressed the PDGF-BB-induced upregulation of proinflammatory mediators (Figure S5F). Together, these in vitro results demonstrate that PDE5A plays a protective role in maintaining the contractile phenotype of VSMCs.

PDE5A Is Required for Exercise-Mediated Protection Against AD

To determine whether PDE5A is essential for exercise-induced aortic protection, we established a BAPN-induced AD mouse model, with mice subjected to treadmill exercise and treated the mice with the PDE5A inhibitor sildenafil (Sild) or vehicle (Veh) control (Figure 4A). Sildenafil administration did not significantly affect SBP in mice (Figure S6A). In vehicle-treated mice, exercise significantly attenuated BAPN-induced AD progression, as evidenced by reduced mortality, decreased maximal aortic diameter, lower AD incidence and rupture rates, reduced expression of proinflammatory mediators (TNF-α, IL [interleukin]-1β, IL-6, and MMP9 [matrix metalloproteinase 9]), improved aortic wall integrity, attenuated collagen deposition, preserved expression of contractile markers (MYH11, α-SMA, and CNN1), and reduced expression of the synthetic marker OPN (Figure 4B through 4J; Figure S6B and S6C). In contrast, pharmacological inhibition of PDE5A with sildenafil completely abolished the protective effects of exercise. Sildenafil-treated exercised mice exhibited increased mortality, enlarged aortic diameter, higher AD incidence and rupture rates, exacerbated medial disruption and elastin degradation, and increased collagen deposition, as well as promoted VSMC phenotypic switching and augmented inflammatory responses (Figure 4B through 4J; Figure S6B and S6C). These mice mirrored the pathological severity observed in sedentary BAPN-treated mice. Collectively, these results demonstrate that PDE5A is necessary for exercise-conferred attenuation of AD.

Figure 4.

Pharmacological inhibition of PDE5A (phosphodiesterase 5A) abrogates the protective effect of exercise in β-aminopropionitrile (BAPN)–induced mice. Mice receiving BAPN water were subjected to 12 weeks of either treadmill exercise (Run) or sedentary (Sed) conditions, while concurrently receiving intraperitoneal injection of sildenafil (Sild) or vehicle (Veh) treatment for 12 weeks (n=23–28). A, Schematic illustration of the experimental timeline. B, Survival curves for BAPN-induced mice undergoing Run or Sed with or without sildenafil treatment. Survival data were analyzed by the Kaplan-Meier method and compared using log-rank tests (n=23–28). C, Representative images of aortas from BAPN-induced mice undergoing Run or Sed combined with sildenafil treatment or not. D, Maximal thoracic aorta diameter in mice receiving BAPN water (n=8). E, The incidence of aortic dissection formation in mice from the indicated experimental groups (n=23–28). F, The incidence of aortic rupture in mice from indicated experimental groups (n=23–28). G and H, Representative hematoxylin and eosin (H&E), elastic Verhoeff-Van Gieson (EVG), and Masson trichrome staining of the thoracic aortas in mice receiving BAPN-supplemented water (G), and quantification of elastin degradation based on a 4‐point scoring system (H; n=6). Scale bar=100 µm. I and J, Western blot analysis (I) and quantification (J) of MYH11 (myosin heavy chain 11), CNN1 (calponin 1), α-SMA (α‐smooth muscle actin), and OPN (osteopontin) protein levels in BAPN-induced mice undergoing Run or Sed combined with sildenafil treatment or not (n=6). Data are presented as mean±SEM. Two-way ANOVA followed by Tukey multiple-comparison test was used in D, H, and J, Fisher exact test was performed in E and F. Created in BioRender. Mao, J. (2026) https://BioRender.com/qlycz8m.

VSMC-Specific Overexpression of PDE5A Attenuates AD Progression

To further evaluate the potential of PDE5A, we constructed an AAV for VSMC-specific overexpression of Pde5a (AAV-Pde5a) and confirmed efficient transduction and expression in VSMCs (Figure S7A through S7D). There was no significant difference in SBP between the AAV-Pde5a-treated mice and AAV-null-injected controls (Figure S7E). Mice injected with AAV-Pde5a or control AAV-null were subjected to BAPN-induced AD (Figure 5A). Under equivalent BAPN challenge, AAV-Pde5a-treated mice exhibited significantly improved survival (2 deaths/25 versus 11 deaths/25 in AAV-null, P<0.05; Figure 5B), along with reduced maximal aortic diameter, decreased AD incidence, and lower aortic rupture rates (Figure 5C through 5F). Histological assessment via hematoxylin and eosin, elastin Van Gieson, and Masson staining revealed attenuated medial disruption, reduced elastin fragmentation, and diminished collagen deposition in AAV-Pde5a-treated mice (Figure 5G and 5H). Immunoblot analysis confirmed that PDE5A overexpression retained contractile marker expression (MYH11, α-SMA, CNN1) and reduced OPN levels (Figure 5I and 5J). Moreover, PDE5A overexpression decreased genes associated with VSMC phenotypic switching and suppressed the expression of proinflammatory mediators (TNF-α, IL-1β, IL-6, and MMP9; Figure S7F; Figure 7G). Together, these findings indicate that VSMC-specific PDE5A overexpression is sufficient to mitigate AD progression, highlighting its role as a central protective mediator.

Figure 5.

Overexpression of PDE5A (phosphodiesterase 5A) in vascular smooth muscle cells (VSMCs) rescues β-aminopropionitrile (BAPN)–induced aortic dissection in mice. Two-week-old male mice were intravenously injected with recombinant adeno-associated virus (AAV) serotype 9 (AAV9) carrying either a control vector or Pde5a-encoding construct. After 1 week, mice received BAPN water for 28 days. A, Schematic illustration of the experimental timeline. B, Survival curves for mice receiving normal water or BAPN water after injection with adeno-associated virus (AAV)-Pde5a or control AAV (AAV-null). Survival data were analyzed by the Kaplan-Meier method and compared using log-rank tests (n=10–25). C, Representative images of aortas from the indicated experimental groups. D, Maximal thoracic aorta diameter in mice from the indicated experimental groups (n=8). E, The incidence of aortic dissection formation in mice from the indicated experimental groups (n=10–25). F, The incidence of aortic rupture in mice from indicated experimental groups (n=10–25). G and H, Representative Hematoxylin and Eosin (H&E), elastic Verhoeff-Van Gieson (EVG), and Masson trichrome staining of the thoracic aortas in mice with injection of AAV-Pde5a or AAV-null (G), and quantification of elastin degradation based on a 4‐point scoring system (H; n=6). Scale bar=100 µm. I and J, Western blot analysis (I) and quantification (J) of MYH11 (myosin heavy chain 11), CNN1 (calponin 1), α-SMA (α‐smooth muscle actin), and OPN (osteopontin) protein levels in mice injected with AAV-Pde5a or AAV-null (n=6). Data are presented as mean±SEM. Two-way ANOVA followed by Tukey multiple-comparison test was used in D, H, and J, Fisher exact test was performed in E and F. Panel A created in BioRender. Mao, J. (2026) https://BioRender.com/qlycz8m.

Figure 7.

Pharmacological inhibition of RUNX1 (runt-related transcription factor 1) with Ro5-3335 attenuates aortic dissection via PDE5A (phosphodiesterase 5A). Three-week-old male mice received β-aminopropionitrile (BAPN) water (1 g/kg per day) or normal drinking water and were subcutaneously injected with Ro5-3335 (20 mg/kg) or vehicle for 28 days. A, Schematic illustration of the experimental timeline. B, Survival curves for normal standard drinking water or BAPN water mice administered with Ro5-3335 or vehicle. Survival data were analyzed by the Kaplan-Meier method and compared using log-rank tests (n=10–27). C, Representative images of aortas in mice across experimental groups. D, Maximal thoracic aorta diameter in mice administered with Ro5-3335 or vehicle (n=8). E, The incidence of aortic dissection formation in mice from indicated experimental groups (n=10–27). F, The incidence of aortic rupture in mice from indicated experimental groups (n=10–27). G and H, Representative hematoxylin and eosin (H&E), elastic Verhoeff-Van Gieson (EVG), and Masson trichrome staining of the thoracic aortas in mice across experimental groups (G), and quantification of elastin degradation based on a 4‐point scoring system (H; n=6). Scale bar=100 µm. I and J, Western blot analysis (I) and quantification (J) of PDE5A, MYH11 (myosin heavy chain 11), CNN1 (calponin 1), α-SMA (α‐smooth muscle actin), and OPN (osteopontin) protein levels in mice across experimental groups (n=6). Data are presented as mean±SEM. Two-way ANOVA followed by Tukey multiple-comparison test was used in D, H, and J, Fisher exact test was performed in E and F. Panel A created in BioRender. Mao, J. (2026) https://BioRender.com/qlycz8m.

RUNX1 Represses PDE5A Transcription in AD

After establishing the regulatory role of PDE5A in VSMC phenotypic transition, we sought to identify upstream factors responsible for its reduced expression during AD. Using a bioinformatic approach, we screened the PDE5A promoter using JASPAR, Cistrome DB, and CIS-BP databases. Integrative analysis of DEGs from mouse aorta identified RUNX1, JUN, and the VDR (vitamin D receptor) as potential transcription factors binding to the PDE5A promoter (Figure 6A). We then examined the effect of exercise on these candidates and observed that BAPN+Run significantly reduced Runx1 mRNA levels, with no significant changes in Vdr or Jun (Figure 6B). Consistently, BAPN treatment increased RUNX1 protein expression in mouse aortas, while exercise counteracted this upregulation (Figure 6C and 6D). Analogously, PDGF-BB stimulation elevated RUNX1 levels in vitro (Figure S8A and S8B). Importantly, RUNX1 was significantly upregulated in human AD tissues (Figure 6E and 6F), suggesting a potential role in PDE5A repression.

Figure 6.

RUNX1 (runt-related transcription factor 1) transcriptionally regulates PDE5A (phosphodiesterase 5A) and modulates phenotypic switching in vascular smooth muscle cells (VSMCs). A, Prediction of transcription factors binding to the Pde5a promoter using JASPAR, Cistrome DB, and CIS-BP databases. The promoter was defined as the region from −2000 bp to +100 bp relative to the transcription start site. A Venn diagram shows overlap between predicted factors and differentially expressed genes (DEGs) in mouse aortic dissection (AD). B, Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis of Runx1, Vdr, and Jun mRNA levels in BAPN-induced mice with or without treadmill exercise (n=4). C and D, Western blot analysis (C) and quantification (D) of RUNX1 protein levels in BAPN-induced mice under either forced treadmill exercise (Run) or sedentary (Sed) conditions (n=6). E and F, Western blot analysis (E) and quantification (F) of RUNX1 protein levels in distal nonlesion tissue (AD-distal non-lesion; n=8) and lesion tissue (AD-lesion; n=8) from patients with AD. G, qRT-PCR analysis of Runx1b and Runx1c mRNA levels in mice across experimental groups (n=6). H through K, Rat aortic smooth muscle cells (RASMCs) were serum-starved for 24 hours, pretreated with Ro5-3335 (20 μmol/L) for 2 hours, and stimulated with platelet-derived growth factor-BB (PDGF-BB, 20 ng/mL) for 48 hours. H, Chromatin immunoprecipitation (ChIP) assay was performed with IgG or anti-RUNX1 antibody, followed by qRT-PCR of the Pde5a promoter (n=3). I, qRT-PCR analysis of Pde5a mRNA levels in RASMCs (n=3). J and K, Western blot analysis (J) and quantification (K) of PDE5A protein levels in RASMCs treated with Ro5-3335 and PDGF-BB (n=4). Data are presented as mean±SEM. Student t test was performed in B and F, 2-way ANOVA followed by Tukey multiple-comparison test was used in D, G, H, I, and K.

RUNX1 functions through 3 major isoforms (RUNX1a/b/c), among which RUNX1a is not expressed in mice due to evolutionary splice site divergence,²⁶ leaving RUNX1b and RUNX1c as the predominant isoforms. To determine which isoform responds to exercise, we designed isoform-specific primers (Figure S8C). Notably, only Runx1b mRNA was significantly elevated by BAPN and suppressed by exercise, identifying RUNX1B as the functionally relevant isoform (Figure 6G). To establish direct transcriptional regulation, we performed chromatin immunoprecipitation coupled with qRT-PCR, which confirmed RUNX1 binding to the PDE5A promoter, an interaction enhanced by PDGF-BB and attenuated by RUNX1 inhibition with Ro5-3335 (Figure 6H). Functionally, Ro5-3335 elevated PDE5A expression at both mRNA and protein levels in PDGF-BB-treated RASMCs (Figure 6I through 6K). Critically, RUNX1 blockade abolished PDGF-BB-induced phenotypic switching (Figure S8D through S8F) and suppressed PDGF-BB-induced cell proliferation (Figure S8G). Additionally, Ro5-3335 significantly mitigated the increase of proinflammatory mediators triggered by PDGF-BB (Figure S8H). These results demonstrate that exercise suppresses RUNX1, particularly the Runx1b isoform, thereby alleviating its repression of PDE5A and attenuating pathological VSMC transition.

RUNX1 Inhibition Protects Against BAPN-Induced AD in Mice

To evaluate the potential of RUNX1 inhibition in AD, we administered Ro5-3335, a benzodiazepine that disrupts RUNX1-CBFβ interactions and reduces RUNX1 activity, to mice following the experimental timeline outlined in Figure 7A. SBP was not significantly altered by Ro5-3335 treatment (Figure S9A). Ro5-3335 treatment significantly improved survival in BAPN-challenged mice, with 12 deaths out of 27 in the BAPN+vehicle group compared with 3 deaths out of 24 in the BAPN+Ro5-3335 group (Figure 7B). Consistently, Ro5-3335 markedly attenuated aortic dilation, as shown by reduced maximal thoracic aortic diameter (Figure 7C and 7D), and significantly decreased both the incidence of AD and aortic rupture (Figure 7E and 7F). Histological evaluation revealed that Ro5-3335 reduced medial thickening, mitigated elastin fragmentation, and diminished collagen deposition (Figure 7G and 7H). At the molecular level, Western blot and immunofluorescence analyses confirmed that Ro5-3335 mitigated VSMC phenotypic switching, along with increased PDE5A expression (Figure 7I and 7J; Figure S9B through S9D). In addition, Ro5-3335 treatment similarly attenuated BAPN-induced inflammation in mice (Figure S9E). Together, these data clearly demonstrate that pharmacological inhibition of RUNX1 confers an attenuating effect on AD development.

Discussion

This study identifies exercise as a novel intervention that attenuates AD, mediated through PDE5A-dependent preservation of VSMC contractile phenotype. Although the involvement of VSMC phenotypic switching in AD is well-established,^2,3 our work extends this knowledge by delineating a specific, exercise-responsive regulatory axis. We demonstrate that AD is associated with downregulation of PDE5A and a pathological phenotypic switch, whereas aerobic exercise upregulates PDE5A, mitigates this switch, and reduces AD severity. Mechanistically, we identify RUNX1 as a key transcriptional repressor of PDE5A, with its upregulation in human AD tissues and BAPN-treated mice, and suppression by exercise establishing a direct transcriptional link between exercise and PDE5A regulation. Critically, either RUNX1 inhibition or PDE5A overexpression recapitulates the protective effects of exercise, including reduced AD incidence, preserved aortic wall integrity, and maintenance of the VSMC contractile phenotype. These findings not only elucidate the molecular basis for exercise-mediated AD protection but also highlight the preventive and therapeutic potential of targeting the RUNX1/PDE5A axis (Figure 8).

Figure 8.

Aerobic exercise confers aortic protection through the RUNX1 (runt-related transcription factor 1)/PDE5A (phosphodiesterase 5A) axis. Under pathological stimulation inducing aortic dissection (AD), elevated RUNX1b expression suppresses PDE5A transcription, promoting phenotypic switching of vascular smooth muscle cells (VSMCs) and exacerbating AD progression. Conversely, aerobic exercise inhibits RUNX1b transcription, thereby upregulating PDE5A expression to maintain the contractile VSMC phenotype and attenuate AD development. Pharmacological inhibition of RUNX1 with Ro5-3335 similarly attenuates AD by restoring PDE5A expression in VSMCs. Collectively, this schematic summarizes the novel RUNX1-PDE5A axis through which aerobic exercise confers protection against AD. Created in BioRender. Mao, J. (2026) https://BioRender.com/4bb5v8k.

The role of exercise in the management of aortic diseases, particularly AD, has garnered increasing interest given its established benefits in general cardiovascular health.⁴⁰ Existing literature suggests that moderate aerobic exercise is not only safe but potentially protective in models of aortic disease, such as thoracic aortic aneurysms and Marfan syndrome, attenuating aortic root dilation and reducing proteolytic degradation through modulation of MMPs (matrix metalloproteinases).^11,12 Notably, these protective effects are underpinned by exercise-induced systemic metabolic remodeling, including enhanced lipid oxidation, optimized liver and muscle glycogen homeostasis, and improved circulating lipid profiles, which collectively alleviate vascular stress and inflammation.^41,42 Clinical studies further support the feasibility and benefits of structured exercise in improving functional capacity and quality of life following surgical repair of AD.¹⁴ Our preclinical exercise protocol is translatable to human moderate-intensity aerobic activity, an achievable goal for individuals at high risk of AD under medical supervision, whereas strenuous activity is contraindicated in acute AD cases. Importantly, we have identified the RUNX1-PDE5A axis in VSMCs as a central mechanism. Exercise suppresses RUNX1, alleviating its repression of PDE5A, which in turn preserves the contractile VSMC phenotype, inhibits synthetic transformation, and reduces medial degeneration and AD incidence. This mechanism aligns with reports that early intervention yields greater benefit,¹¹ underscoring the importance of timing. Notably, the RUNX1-PDE5A pathway represents a targetable mechanism that may explain the conserved benefits of exercise across different aortic disease models. Our finding that PDE5A overexpression or RUNX1 inhibition phenocopies exercise’s effects further highlights its preventive potential, suggesting preventive strategies for patients unable to engage in intense physical activity.

As a key cGMP-specific phosphodiesterase, PDE5A plays a critical role in regulating VSMC homeostasis, with profound implications for AD pathogenesis.^20,21,43 PDE5A hydrolyzes cGMP, a second messenger that promotes VSMC contractility through activation of PKG (protein kinase G) and maintenance of contractile protein expression.^44,45 Our findings establish that PDE5A expression is downregulated in VSMCs in both human AD tissues and murine models, coinciding with pathological VSMC phenotypic switching.²¹ This downregulation is functionally critical, as genetic evidence from VSMC-specific Pde5a knockout mice demonstrates that PDE5A deficiency exacerbates aortic aneurysm and dissection, whereas its overexpression is protective.²⁰ Thus, the preventive induction of PDE5A in VSMCs, whether through exercise-induced upregulation, targeted overexpression, or as evidenced by genetic models, mitigates phenotypic switching and reduces AD incidence, highlighting its essential causal role. The role of PDE5A appears to be context-dependent. For example, Ang II (angiotensin II) can transiently upregulate PDE5A to drive acute VSMC proliferation,⁴³ while its chronic downregulation in AD disrupts contractile phenotype maintenance. Similarly, PDE5A inhibition may have short-term vasodilatory effects,¹⁸ but its chronic loss-of-function drives AD pathogenesis. Collectively, these findings from pharmacological and genetic knockout models firmly position PDE5A as a pivotal, context-dependent regulator of VSMC phenotype in AD, with its preservation representing a promising preventive strategy to stabilize the aortic wall.

RUNX1 is a hematopoietic transcription factor with critical roles in hematopoiesis, operating through 3 major isoforms (RUNX1a/b/c).^24,26 Although RUNX1b and RUNX1c contain conserved DNA-binding and transactivation domains, the truncated RUNX1a isoform is primate-specific and absent in mice, conferring distinct transcriptional outputs.²⁶ In VSMCs, RUNX1 has emerged as a key regulator of pathological remodeling, implicated in promoting inflammation, proliferation, and phenotypic switching.^28,46,47 Our work identifies RUNX1, particularly the predominant RUNX1B isoform, as a pivotal driver of AD pathogenesis by directly repressing PDE5A transcription in VSMCs, thereby triggering phenotypic switching and medial degeneration. This contrasts with a report suggesting RUNX1 activation inhibits phenotypic switching in an Ang II–induced aortic aneurysm,²⁸ a discrepancy likely attributable to isoform-specific functions or divergent disease contexts. Our findings align with other studies demonstrating RUNX1 upregulation in pathological VSMCs and its role in promoting a proliferative, synthetic phenotype.⁴⁸ These findings establish RUNX1 as a preventive and therapeutic target in AD. Future studies should delineate how upstream signals, including exercise and hemodynamic stress, regulate RUNX1 to maintain aortic integrity, which is essential for harnessing its potential in AD prophylaxis.

The BAPN-induced AD model used in this study is based on the specific inhibition of LOX (lysyl oxidase), which disrupts the crosslinking of collagen and elastic fibers, thereby reducing the structural integrity of the aortic wall and increasing its fragility.^49,50 This ECM disruption actively drives VSMCs toward a pathological synthetic phenotype, creating a vulnerability that may predispose to rupture rather than gradual dilation, with susceptibility influenced by specific factors, such as age, sex, and ECM integrity. Notably, in contrast to this model of ECM-driven fragility, the human AD tissues analyzed in our study were predominantly from cases of hypertension-associated dissection. Whereas hypertension imposes primarily a hemodynamic insult and BAPN induces a structural matrix defect, both conditions converge on a dysregulated RUNX1-PDE5A axis and a pathological VSMC phenotypic switch. This convergence suggests that this pathway may act as a common downstream effector mechanism in AD pathogenesis, bridging distinct initial triggers. Furthermore, the ascending and descending aortas may respond differently to BAPN, displaying distinct pathological features.⁵¹ Our study identifies an exercise-responsive, cell-autonomous defense mechanism against this fragility. We demonstrate that the RUNX1-PDE5A axis preserves the VSMC contractile phenotype, thereby enhancing the functional resilience of the aortic wall even when its structural scaffold is compromised. This mechanism increases the wall’s intrinsic strength, which is fundamental to tolerating hemodynamic stress-the ultimate trigger for AD.^52,53 The protection observed in our normotensive model suggests this pathway strengthens the aorta independent of blood pressure-lowering effects. Intriguingly, our blood pressure measurements revealed a modest downward trend in SBP among the exercised mice towards the later observation time points. However, this did not reach statistical significance within the timeframe of our study and does not alter our primary conclusion of a blood pressure-independent, VSMC-autonomous protective mechanism. Conversely, the loss of protection with the PDE5A inhibitor sildenafil reveals a complex interplay, where potential afterload reduction may be counteracted by the disruption of this crucial VSMC-stabilizing pathway. Thus, targeting the RUNX1-PDE5A axis may represent a promising preventive strategy to augment aortic wall resistance in conditions of underlying ECM weakness, such as heritable aortopathies.⁴⁹ Future validation in hypertensive models will be invaluable to translate these cell-autonomous insights into a more comprehensive understanding of AD prevention.

Although this study identifies the RUNX1-PDE5A axis as a key downstream mechanism, the upstream signals linking exercise to this pathway warrant consideration. We propose that the benefit likely stems from the integrated effects of multiple exercise-induced adaptations, which converge to suppress pathological RUNX1 activation in VSMCs. First, exercise-mediated systemic metabolic improvement, marked by enhanced insulin sensitivity and altered circulating metabolites, reduces chronic low-grade inflammation and oxidative stress, both of which are potent inducers of RUNX1.⁵⁴ Notably, this mitigation of inflammatory tone may also underpin the protective effect of the RUNX1 inhibitor Ro5-3335, positioning RUNX1 itself as a key transcriptional node linking metabolic stress to vascular inflammation and phenotypic switching.⁵⁵ Second, exercise alters hemodynamic forces on the aortic wall, including shear stress and cyclic stretch, which are fundamental regulators of VSMC phenotype via mechanosensitive pathways.^56,57 Third, contracting skeletal muscle releases myokines (eg, irisin) that may exert endocrine effects on the vasculature.⁵⁸ Together, these signals presumably create a microenvironment that antagonizes RUNX1 upregulation, thereby preserving PDE5A expression and VSMC contractility. Defining the predominant upstream trigger(s) is a crucial focus for future research aimed at developing exercise-mimetic preventive strategies.

In summary, this study reveals a previously unknown mechanism through which exercise attenuates AD. Exercise inhibits RUNX1, which then upregulates PDE5A and preserves the contractile phenotype of VSMCs. Specifically, pharmacological inhibition of RUNX1 or PDE5A overexpression through AAV-mediated overexpression of PDE5A successfully preserved the VSMC contractile phenotype under AD stress and reduced AD incidence, mirroring the protective effects of exercise. Therefore, targeting the RUNX1-PDE5A axis represents a promising strategy for AD prevention.

ARTICLE INFORMATION

Acknowledgments

The authors thank the Core Facility of the Research Center of Basic Medical Sciences at Tianjin Medical University for technical support. Figures 1D, 3A, 4A, 5A, 7A, and 8 and the graphic abstract were created using BioRender.com.

Disclosures

None.

Supplemental Material

Tables S1–S3

Figures S1–S9

Major Resources Table

Supplementary Material

atv-46-e324293-s001.pdf ^{(2MB, pdf)}

atv-46-e324293-s002.pdf ^{(3.8MB, pdf)}

Funding Statement

This research was supported by the National Natural Science Foundation of China (82370299 and 82570343), the Natural Science Foundation of Tianjin Municipal Science and Technology Commission (23JCYBJC00280).