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. 2025 Aug 29;20(8):e0322434. doi: 10.1371/journal.pone.0322434

β-aminopropionitrile-induced thoracic aortopathy is refractory to cilostazol and sildenafil in mice

Samuel C Tyagi 1,2,3,4, Sohei Ito 1,2, Jacob C Hubbuch 1,2,3,4, Michael K Franklin 1,2, Deborah A Howatt 1,2, Hong S Lu 1,2,3, Alan Daugherty 1,2,3,*, Hisashi Sawada 1,2,3,*
Editor: Jeffrey S Isenberg5
PMCID: PMC12396674  PMID: 40880357

Abstract

Thoracic aortopathies are life-threatening diseases including aneurysm, dissection, and rupture. Cilostazol, a phosphodiesterase (PDE) 3 inhibitor, and sildenafil, a PDE5 inhibitor, have been used clinically for peripheral arterial disease and erectile dysfunction or pulmonary hypertension, respectively. Recent studies report their effects on abdominal aortic aneurysm formation. However, their impacts on thoracic aortopathy remain unknown. In this study, we investigated whether cilostazol and sildenafil affect thoracic aortopathy induced by β-aminopropionitrile (BAPN) administration in mice. Bulk RNA sequencing analysis revealed that BAPN administration upregulated Pde3a transcription in the ascending aorta and Pde5a in both ascending and descending regions before thoracic aortopathy formation. Next, we tested the effects of cilostazol or sildenafil on BAPN-induced thoracic aortopathy. BAPN-administered mice were fed a diet supplemented with either cilostazol or sildenafil. Mass spectrometry measurements determined the presence of cilostazol or sildenafil in the plasma of mice fed drug-supplemented diets. However, neither drug altered BAPN-induced aortic rupture nor aneurysm formation and progression. These results provide evidence that cilostazol and sildenafil did not influence BAPN-induced thoracic aortopathy in mice.

Introduction

Thoracic aortopathy is a spectrum of diseases, including aneurysm, dissection, and rupture. A key pathological feature of thoracic aortopathy is extracellular matrix (ECM) remodeling, including elastic fiber fragmentation and excessive collagen fiber deposition [1]. Given the vital role of ECM in maintaining structural integrity, preventing ECM changes may inhibit development and progression of thoracic aortopathy. Among available mouse models, β-aminopropionitrile (BAPN) administration is increasingly used in preclinical studies [2–6]. BAPN inhibits elastin and collagen crosslinking by blocking lysyl oxidase activity, leading to ECM changes that are a ubiquitous feature of thoracic aortopathy. Therefore, BAPN administration may be an approach to investigate mechanisms and therapeutic potential of thoracic aortopathy.

Phosphodiesterases (PDEs) regulate intracellular cyclic nucleotide abundance by catalyzing their hydrolysis into inactive forms [7,8]. Within the PDE family, PDE3 and PDE5 have garnered interest in their potential roles in the pathophysiology of aortic diseases [9–13]. Cilostazol, a PDE3 inhibitor used clinically for peripheral arterial disease, has vasodilatory and antiplatelet effects [14]. A recent preclinical study demonstrated that cilostazol attenuated angiotensin II-induced abdominal aortic aneurysms in hypercholesterolemic mice [9]. In contrast, sildenafil, a PDE5 inhibitor used for erectile dysfunction and pulmonary hypertension, exacerbated abdominal aortic aneurysm induced by a combination of BAPN oral administration and elastase topical application in normolipidemic mice [13]. These findings provide evidence that cilostazol and sildenafil have differential effects on abdominal aortic aneurysms. However, it remains unknown whether these drugs affect thoracic aortopathy formation and progression. The mechanisms underlying aortopathy differ between the thoracic and abdominal regions [15], highlighting a need for preclinical studies to determine the effects of these drugs on thoracic aortopathy.

Based on clinical and preclinical evidence, we hypothesized that cilostazol attenuates thoracic aortopathy, whereas sildenafil exacerbates it. In this study, we examined the effects of cilostazol and sildenafil on thoracic aortopathy associated with changes in ECM. BAPN-administered mice were fed a standard laboratory diet or a diet supplemented with cilostazol or sildenafil, and thoracic aortopathy formation and progression were compared across groups.

Materials and methods

Mice

C57BL/6J mice at 3 weeks of age were purchased from The Jackson Laboratory (#000664) and housed in individually ventilated cages (5 mice/cage) under a 14-hour light and 10-hour dark cycle. Teklad Sani-Chip bedding (#7090A, Inotiv) was used for cage bedding. Mice were fed a standard rodent laboratory diet (#2918, Inotiv), cilostazol-supplemented diet (0.315 g/kg, #TD.190797, Teklad), or sildenafil-supplemented diet (0.315 g/kg, #TD.190796, Teklad) (Fig 1). Five days after initiating dietary interventions, BAPN (0.5% wt/vol, #A3134-25G, Millipore-Sigma or #A0796-500G, TCI Co.) was administered in drinking water. Our previous study revealed that BAPN administration for 28 days induced acute aortic pathologies, and 84-day administration results in progressive vascular remodeling. Therefore, BAPN was administered for 28 and 84 days to evaluate the effects of drugs on both the initiation and progression of thoracic aortopathy, respectively [6]. BAPN-induced thoracic aortopathy does not display significant sexual dimorphism [6]. Therefore, the present study evaluated the effects of PDE inhibition in male mice. Based on our previous study [6], the present study was designed with a sample size of n = 15 per group to achieve a statistical power greater than 0.8 to detect a 1.1 mm difference with a standard deviation of 0.9, assuming an alpha level of 0.05. Mice were randomly assigned to the study groups, and no mice were excluded from the study. Drinking water was replaced twice weekly. All animal experiments adhered to the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines and were approved by the University of Kentucky Institutional Animal Care and Use Committee (2018–2967).

Fig 1. Scheme for experimental designs.

Fig 1

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BAPN was administered to male C57BL/6J mice fed a normal, cilostazol-, or sildenafil-supplemented diet for either 4 or 12 weeks.

Determination of aortic rupture and dimensions

All study mice were monitored at least once daily. Necropsies were performed immediately upon discovery of deceased mice to determine the cause of death. Aortic rupture was defined by the presence of extravascular blood accumulated in the thoracic or abdominal cavity. The site of blood egress was identified based on the location of the blood clot and a discernible disruption of the aortic wall. Mice were euthanized via intraperitoneal injection of ketamine (90 mg/kg, #11695-6840-1, Covetrus) and xylazine (10 mg/kg, #11695-4024-1, Covetrus) cocktail at 28 or 84 days following BAPN administration. The right atrial appendage was excised, and saline (~10 mL) was perfused via the left ventricle. After removing periaortic tissues, a black plastic sheet was inserted underneath the aorta [5,16]. A ruler was placed adjacent to the aorta for measurement calibration. Then, in situ aortic images were captured using a stereoscope (SMZ25, Nikon). Aortic diameters were measured perpendicularly to the aortic axis at the most dilated area of ascending and descending regions using NIS-Elements AR software (v5.11, Nikon). Measurements were verified by an individual who was blinded to the identity of the study groups.

Mass spectrometry

Plasma cilostazol and sildenafil concentrations were measured using mass spectrometry in the Research Mass Spectrometry and Proteomics Core at the University of Kentucky. Sildenafil (1 mg/mL in solvent, Cerilliant S-010), cilostazol (powder, Sigma PHR1503-1G), and d8-sildenafil (1 mg/mL, LGC TRC-5435003) standards were used. Solvents included LC-MS grade methanol (Millipore MX0486), methyl tert-butyl ether (MTBE, Fisher E127), LCMS-Water, formic acid, and ammonium formate. Drug-free serum was used as the biological matrix. Chromatographic separation was performed on an ACQUITY UPLC BEH C18 column [130Å, 50 mm x 2.1 mm, 1.7 µm particle size] (Waters Part# 186002350).

Sildenafil and cilostazol were extracted from serum via liquid-liquid extraction. Briefly, serum (100 µL) was spiked with appropriate standards or methanol (for negative controls). Internal standard (d8-sildenafil, 100 µL at 80 ng/mL) was added to all samples. Samples were extracted with methyl tert-butyl ether (MTBE;5 mL) by roto-racking for 5 minutes, and centrifugation at 2,500 rpm (~1000 x g) for 5 minutes. The MTBE layer was transferred to a clean tube and evaporated to dryness under nitrogen at 40°C. Analytes were reconstituted in 100 µL of mobile phase A (85% vol/vol) and mobile phase B (15% vol/vol). Mobile phase A was 5 mM ammonium formate in formic acid (0.01% vol/vol) in water, and mobile phase B was 100% methanol. Resuspended samples were transferred to silanized vials for LC-MS/MS analysis. Calibrators were prepared by adding sildenafil and cilostazol to drug-free serum to achieve concentrations ranging from 1 to 200 ng/ml for each analyte. Quality control samples contained sildenafil and cilostazol at 60 ng/mL, respectively.

Extracted samples were analyzed on a Thermo Scientific TSQ Altis Plus Triple Quadrupole Mass Spectrometer coupled to a Vanquish Flex liquid chromatography system. Chromatographic separation was performed on an ACQUITY UPLC BEH C18 column (50 mm x 2.1 mm, 1.7 µm particle size; Waters). Mobile phases were: A, 5 mM ammonium formate in formic acid (0.1% vol/vol) in water; and B, methanol (100% vol/vol). Compounds were separated across a linear gradient from 1% mobile phase B to 100% mobile phase B over 5 minutes. The flow rate was set to 0.5 ml/min. The column temperature was set to 40°C. Detection was performed via multiple reaction monitoring (MRM) in positive electrospray ionization mode. Instrument parameters were set to the following: positive spray voltage 3000 V, sheath gas: 50, aux gas: 1, sweep gas: 1, ion transfer tube temperature: 325°C, vaporizer temperature: 350°C, Q1 resolution was set to 0.7 (FWHM), Q3 resolution was set to 1.2 (FWHM), CID gas (mTorr): 1.5.

RNA sequencing data analysis

Read count data were obtained from our previous data posted on the Genome Expression Omnibus (GSE241968, RRID:SCR_005012) [6] and normalized using the “trimmed mean of M values” method in edgeR (v3.36.0, RRID:SCR_012802) on R (v4.1.0). Subsequently, normalized read counts for Pde3a and Pde5a were extracted. In the sequencing, aortic samples were harvested from ascending and descending regions at 1 week of BAPN administration, corresponding to the pre-pathological phase of thoracic aortopathy. Read count data of Pde3a and Pde5a were extracted and compared between vehicle- and BAPN-administered mice in ascending and descending regions. The R code used in this study is available upon request.

Statistical analyses

Data are represented as individual data points with the median and 25th/75th percentiles. TMM-normalized read count data were analyzed by Student’s t-test in ascending and descending regions separately. Log-Rank test was used to compare survival rates between groups in Kaplan-Meier curves. Normality and homogeneity of variance were assessed by Shapiro-Wilk and Brown-Forsythe tests, respectively. One-way analysis of variance followed by Holm-Sidak test was performed for parametric comparisons, whereas Kruskal-Wallis followed by Dunn’s post-hoc test was performed for non-parametric comparisons. P < 0.05 was considered statistically significant. Statistical analyses were performed using SigmaPlot version 15.0 (SYSTAT Software Inc., RRID:SCR_003210).

Results

Pde3a and Pde5a mRNA were upregulated in the pre-pathological phase of the thoracic aorta in BAPN-administered mice

We first investigated the transcriptomic changes of aortic PDE3 and PDE5 in BAPN-administered mice using our previous bulk RNA-sequencing data [6]. While no statistical difference was observed in the descending region, Pde3a mRNA abundance was increased significantly in the ascending aorta of BAPN-administered mice compared to vehicle-administered mice (Fig 2A). Pde5a mRNA abundance was increased in both regions of BAPN-administered mice (Fig 2B). These data demonstrated upregulation of aortic PDE3 and PDE5 in the prepathological phase of BAPN-administered mice.

Fig 2. Increase of Pde3a and Pde5a mRNA abundance in the thoracic aorta of BAPN-administered mice.

Fig 2

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Normalized read count data from bulk RNA sequence data (GSE241968) for (A) Pde3 and (B) Pde5 mRNA in ascending (Asc) and descending (Dsc) aortas of mice administered with either vehicle or BAPN for 1 week. P values were determined by Student’s t-test (A) or Mann-Whitney test (B) in each region.

Verification of cilostazol and sildenafil administration in mice fed drug-supplemented diet

To determine the role of PDE3 and PDE5 in development of BAPN-induced thoracic aortopathy, BAPN-administered mice were fed a diet supplemented with either cilostazol or sildenafil. A standard laboratory diet was administered to the BAPN-administered mice as a control group (Fig 1). After 28 days of BAPN administration, mice were euthanized. Mass spectrometry analysis was performed on plasma samples of these mice as well as those on a standard diet to validate the presence of these drugs in plasma. As expected, neither cilostazol nor sildenafil was detected in the plasma of mice fed a standard laboratory diet (Fig 3A, B). The cilostazol-supplemented diet increased plasma cilostazol concentrations, while the sildenafil-supplemented diet increased only sildenafil concentrations (Fig 3A, B). These data indicate the effective delivery of drugs using diet supplementation in BAPN-administered mice.

Fig 3. Determination of plasma concentrations of cilostazol and sildenafil in mice fed drug-supplemented diet.

Fig 3

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Plasma concentrations of (A) cilostazol and (B) sildenafil in mice fed control, cilostazol-, or sildenafil-contained diet. N = 5-9/group. P-values were determined by Kruskal-Wallis test followed by Dunn’s test.

Cilostazol and sildenafil did not affect BAPN-induced thoracic aortopathy formation

Subsequently, aortic phenotypes were evaluated after 28 days of BAPN administration. Consistent with previous reports [6], BAPN (0.5% wt/vol) administration resulted in high mortality due to aortic rupture and dissection in C57BL/6J mice. Aortic rupture-related death was observed in 44% of control mice during BAPN administration (Fig 4A). Although the mass spectrometry analysis validated the presence of cilostazol and sildenafil in the plasma (Fig 3A, B), cilostazol or sildenafil supplementation did not change the survival rate compared to mice fed control diet (Fig 4A). Maximum thoracic aortic diameters, measured using in situ aortic images, were comparable among three groups regardless of aortic regions (Fig 4B–D).

Fig 4. Cilostazol or sildenafil did not change BAPN-induced thoracic aortopathy after 28 days of administration.

Fig 4

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(A) Survival rate, (B) gross appearance of thoracic aortas, and aortic diameters in (C) ascending and (D) descending regions of control, cilostazol, or sildenafil-administered mice. P values were calculated by Log-Rank for (A) or Kruskal Wallis tests for (C, D).

Cilostazol and sildenafil did not affect BAPN-induced thoracic aortopathy progression

We then extended the study duration to 12 weeks to examine the effects of cilostazol and sildenafil on the progression of BAPN-induced thoracic aortopathy. Prolonged BAPN administration resulted in 68% mortality due to aortic rupture or dissection (Fig 5A). Similar to findings with 4 weeks of BAPN administration, cilostazol- and sildenafil-supplemented diets did not affect survival rates during 12 weeks of BAPN administration (Fig 5A). In situ aortic measurements showed that neither diet prevented nor exacerbated BAPN-induced thoracic aneurysm formation in the ascending or descending aorta after 12 weeks of BAPN administration (Fig 5B–D).

Fig 5. Cilostazol or sildenafil did not suppress the progression of BAPN-induced thoracic aortopathy after 84 days of administration.

Fig 5

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(A) Survival rate, (B) gross appearance of thoracic aortas, and aortic diameters in (C) ascending and (D) descending regions of control, cilostazol, or sildenafil-administered mice. P values were calculated by Log-Rank for (A) or Kruskal Wallis tests for (C, D).

Discussion

In the present study, we found that aortic Pde3a transcription in the ascending region and Pde5a transcription in both ascending and descending regions were upregulated before thoracic aortopathy formation during BAPN administration. Although the presence of cilostazol and sildenafil was detected, neither of the two drugs changed the development and progression of BAPN-induced thoracic aortopathy in mice.

A study reported that cilostazol attenuated angiotensin II-induced abdominal aortic aneurysm in hypercholesterolemic mice [9]. The protective effects of cilostazol were associated with the suppression of inflammatory cytokine expression. Since vascular inflammation is involved in the pathophysiology of BAPN-induced thoracic aortopathy [17], we hypothesized that cilostazol similarly inhibits disease progression in this model. However, in the present study, cilostazol failed to suppress the formation and progression of thoracic aortopathy in BAPN-administered mice. BAPN-induced thoracic aortopathy results from the direct inhibition of lysyl oxidase-mediated ECM maturation, leading to structural destabilization of the aortic wall [6]. Therefore, inflammatory responses in this model appear to be a secondary consequence rather than a primary driver of disease development, which may explain the ineffectiveness of cilostazol in suppressing BAPN-induced thoracic aortopathy.

Sildenafil has been clinically used to treat erectile dysfunction and pulmonary hypertension via its vasodilatory effect through the increase of nitroxide production [18–20]. A previous study found that sildenafil enhanced progression of abdominal aortic aneurysms induced by BAPN administration and topical elastase application [13]. This effect was attributed to impaired aortic contractility, characterized by increased protein kinase G abundance and decreased myosin light chain phosphorylation [13]. However, in our study, sildenafil did not affect the formation and progression of BAPN-induced thoracic aortopathy. Although both studies used BAPN to induce aortopathy, the disease locations differ. There is evidence that aortopathy exhibits regional heterogeneity that are attributable to distinct disease mechanisms [15]. This indicates that BAPN-induced thoracic aortopathy may not be driven by protein kinase G activation and myosin light chain phosphorylation. Moreover, bulk RNA sequencing in our previous study demonstrated that the molecular mechanisms driving BAPN-induced thoracic aortopathy are independent of the transforming growth factor β signaling pathway and the renin-angiotensin system [6], both of which are commonly implicated in aortopathy mechanisms. These results indicate that BAPN-induced thoracic aortopathy is mediated by mechanisms primarily driven by extracellular matrix disruption, distinguishing it from other aortopathy models. Further studies are required to elucidate the precise mechanisms underlying BAPN-induced thoracic aortopathy. In particular, comprehensive histological analyses will be essential to identify the underlying cellular and molecular changes. In addition, single-cell RNA sequencing may provide deeper insights into downstream pathway mechanisms.

Although neither drug affected BAPN-induced thoracic aortopathy formation and progression, measurements of drug concentrations using mass spectrometry confirmed that dietary supplementation with cilostazol and sildenafil increased their plasma concentrations in mice in this study. Throughout the study, mice had continuous access to drug-supplemented diets. However, plasma half-lives of cilostazol and sildenafil in rodents are relatively short (cilostazol: 2–4 hours, sildenafil: 0.3–2 hours, in rats) [21,22]. Therefore, it remains unclear whether their plasma concentrations remained consistently elevated throughout the experiment. In addition, we used a drug concentration of 0.315 g/kg in the food, which corresponds to an estimated drug intake of approximately 1.3 mg/day, based on the commonly reported feeding behavior of C57BL/6J mice (4 g food/day) [23,24]. Previous studies investigating the effects of these drugs in several types of cardiovascular and cerebral diseases, such as heart failure and cognitive dysfunction [25–31]. While many of these studies using sildenafil employed doses around 1.3 mg/day, some studies investigating cilostazol administered higher doses of up to 60 mg/day. Therefore, it remains unknown whether higher doses can attenuate thoracic aortopathy. it remains unknown whether higher doses can attenuate thoracic aortopathy. Future studies are needed to evaluate the effects of continuous administration and higher doses of these drugs on BAPN-induced thoracic aortopathy formation and progression. Also, frequent plasma sampling and assessment of downstream signaling pathways, such as cyclic adenosine or guanosine monophosphates, are important to validate effective cilostazol and sildenafil administrations.

In the present study, BAPN (0.5% wt/vol in drinking water) was used to induce thoracic aortopathy in young male C57BL/6J mice. Importantly, our previous study demonstrated that BAPN concentrations in drinking water is a critical determinant of disease development and severity in this model [6]. While 0.1% BAPN led to minimal pathology and no mortality, increasing the concentration to 0.3% resulted in a significant incidence of aortic rupture (60%), with surviving mice developing thoracic aortopathy. The highest BAPN concentration used (0.5% wt/vol) concentration was even more severe, with an 80% mortality rate due to rupture, and the surviving mice exhibited extensive aortic pathology, including profound dilatation and involvement of the descending thoracic aorta. These findings highlight the dose-dependent effects of BAPN-induced thoracic aortopathy. Based on our previous studies examining the dose-response of BAPN [6], the present study used 0.5% BAPN to ensure a sufficiently high disease incidence that would allow robust assessment of phenotypic outcomes. We acknowledge that this high concentration leads to higher mortality, potentially interfering with data interpretation for the sildenafil group, since we hypothesized that inhibiting PDE5 augments thoracic aortopathy.

In conclusion, this study provides evidence that cilostazol and sildenafil do not influence BAPN-induced thoracic aortopathy in mice.

Supporting information

S1 File. Supplement excel file.

(XLSX)

pone.0322434.s001.xlsx (27KB, xlsx)

Data Availability

All relevant data are within the paper and its Supporting information files.

Funding Statement

The studies reported in this article were supported by the National Heart, Lung, and Blood Institute of the National Institutes of Health (R35HL155649), the American Heart Association (23MERIT1036341, 24CDA1268148), and the Leducq Foundation for the Networks of Excellence Program (Cellular and Molecular Drivers of Acute Aortic Dissections).

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Decision Letter 0

Jeffrey Isenberg

9 May 2025

PONE-D-25-15324β-aminopropionitrile-induced thoracic aortopathy is refractory to cilostazol and sildenafil in micePLOS ONE

Dear Dr. Sawada,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Jun 23 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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PLOS ONE

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Reviewers' comments:

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Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #2: Yes

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The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: In the manuscript titled “β-aminopropionitrile-induced thoracic aortopathy is refractory to cilostazol and sildenafil in mice” the authors test the notion that altering the secondary degradation of cGMP by limiting the PDEs that do this would alert the outcome of vascular degradation in the aorta in mice given β-aminopropionitrile. This agent prevents stability in the elastic fibers that are essential to major artery function and mechanical health. It is a chemical model that produces in short order what occurs with time in some people, namely loss of functional elastic fibers in the aorta. PDE blockers have been in use for cardiovascular issues for years. They target several members of a large class of agents that are a second line of control of essential cGMP, the latter being the major signal agent of vascular nitric oxide generated by endothelial cells. Mice were given two agents, cilostazol or sildenafil in the diet and levels tacked with mass spec. Neither seemed to decrease aortic aneurysms rates.

A version of this manuscript was published on-line as a pre-print: bioRxiv [Preprint]. 2025 Mar 27:2025.03.24.645113.

Novelty: Some work has been done with β-aminopropionitrile in rodents. A PubMed search with the term “β-aminopropionitrile aorta mice” yielded ~130 papers. Indeed, some of this work is from the authors. For example, Arterioscler Thromb Vasc Biol. 2024 Jul;44(7):1555-1569. Of note this latter paper provides details about the murine model. But this raises the question, why the treatment was not applied to mice when they were phenotype analysis for the 2024 paper. While data in both papers seems helpful, I do not understand why the authors did not combine the work into a single paper.

Rationale: The Introduction offers a rationale based on data showing conflicting effects of the two treatments with one limiting aneurysm formation and the other worsening them. Thus, it seems reasonable to consider what accounts for this. However, in truth the models of arterial disease were not the same so that alone might be a reason for using one model and testing both agents.

Methods: The text on the timing of treatments and injury is a little confusing. Also, why give β-aminopropionitrile for 28 or 84 days? What was the reason for looking at plasma levels of the treatments; perhaps as a means to estimate the effective delivered amounts? Did you suspect that mice were not taking the agents by eating less. How many samples over time were taken and how did this compare to the administration of the treatments. I would be surprised that half-lives of these agents are not published for mice and rats. Provide any available data on food and water intake, activity, and weight changes during the study intervals. Justify using just male mice. Provide a power analysis for cohort sizes. Provide any vitals and cardiovascular information on the mice -BP, pulse, cardiac output, ejection fraction, etc. if available. If not give us reasons why this was not checked as a method to ascertain if the agents made any difference globally, and not just at the aorta.

I am concerned about the study design. As the authors published already on the high mortality of the stress (>40%) why go out to the extent that the same was achieved in the present study. This would seem to be unnecessary as one could look at early time points to find if the treatments made any difference.

Data: The whole tissue mRNA data is not too helpful. What should be done is single cell seq followed if indicated with bulk analysis. We just do not what to make of increase PDE mRNA - is it form immune cells, fibroblasts, smooth muscle cells, endothelial cells. They all respond to NO and cGMP and so have counter controls like PDEs. I suggest trying to localize this information. Maybe high contrast and magnification immunofluorescence of the aortic walls would help.

How did you validate the morphometric approaches used on the resected arch and descending aorta? I think in vivo doppler or other non-invasive approaches would be much more helpful here.

The paper has a limit in mechanism. To begin why not map the NO-cGMP-PKG pathway in the samples from the mice; the target might be PKG. As well, while transcripts to several PDEs changed there is no explanation as to why? Was it feedback from a lock of NO-cGMP signal. And what would account for the loss of NO-cGMP or any relative resistance to the same? Did the β-aminopropionitrile cause secondary/tertiary modifications on pathway molecules that altered their function? And/or did it upregulate known inhibitors of the NO-cGMP-PKG cascade such as thrombopsindin-1, superoxide, etc. (see: Proc Natl Acad Sci U S A. 2005 Sep 13;102(37):13141-6 and Nat Rev Cancer. 2009 Mar;9(3):182-94). I would bet a link to β-aminopropionitrile and superoxide is hinted at but not fully defined, while low NO turns on thrombospondin-1 expression (see Proc Natl Acad Sci U S A. 2005 Sep 13;102(37):13147-52).

Legends: Add the exact statistics test used for each graph.

Figures: Ok but was all of the color needed?

Indicate if AI was or was not used in manuscript perpetration and a section on what each author contributed. As well, mention somewhere that the paper is online as a pre-print.

Reviewer #2: This is an automated report for PONE-D-25-15324. This report was solicited by the PLOS One editorial team and provided by ScreenIT.

ScreenIT is an independent group of scientists developing automated tools that analyze academic papers. A set of automated tools screened your submitted manuscript and provided the report below. Each tool was created by your academic colleagues with the goal of helping authors. The tools look for factors that are important for transparency, rigor and reproducibility, and we hope that the report might help you to improve reporting in your manuscript. Within the report you will find links to more information about the items that the tools check. These links include helpful papers, websites, or videos that explain why the item is important. While our screening tools aim to improve and maintain quality standards they may, on occasion, miss nuances specific to your study type or flag something incorrectly. Each tool has limitations that are described on the ScreenIT website. The tools screen the main file for the paper; they are not able to screen supplements stored in separate files. Please note that the Academic Editor had access to these comments while making a decision on your manuscript. The Academic Editor may ask that issues flagged in this report be addressed. If you would like to provide feedback on the ScreenIT tool, please email the team at ScreenIt@bih-charite.de. If you have questions or concerns about the review process, please contact the PLOS One office at plosone@plos.org.

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Reviewer #2: No

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Attachment

Submitted filename: report_10.1101+2025.03.24.645113.pdf

pone.0322434.s002.pdf (598.1KB, pdf)

Decision Letter 1

Jeffrey Isenberg

16 Jul 2025

PONE-D-25-15324R1β-aminopropionitrile-induced thoracic aortopathy is refractory to cilostazol and sildenafil in micePLOS ONE

Dear Dr. Sawada,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================

The authors will note that many details were requested by the Reviewers. As well, some questions will require new data or reanalysis of existing data. 

==============================

Please submit your revised manuscript by Aug 30 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

  • A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

  • A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

  • An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols .

We look forward to receiving your revised manuscript.

Kind regards,

Jeffrey S Isenberg, MD, MPH

Academic Editor

PLOS ONE

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If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise. 

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Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #3: (No Response)

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #3: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #3: No

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

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Reviewer #3: Yes

**********

6. Review Comments to the Author

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Reviewer #3: Comments:

The author did the research on two inhibitor drugs (phosphodiesterase inhibitors), one is cilostazol a PDE3 inhibitor and other is sildenafil a PDE5 inhibitor, to investigate the effect of these two drugs on the development and progression of disease thoracic aortopathy induced by β-aminopropionitrile (BAPN) in mice. Author also did bulk RNA sequencing to see the β-aminopropionitrile (BAPN) effect on Pde3a and Pde5a gene transcription level. Although the author found both genes were upregulated in the disease development but dietary administration of both drugs failed to alter the outcomes i.e. aneurysm formation, aortic rupture, and mortality. Finally author concluded based on the experimental data that cilostazol and sildenafil did not influence BAPN-induced thoracic aortopathy in mice.

The author has investigated a very important disease of the concerned. The study is well structured and addressed an important question. The most interested thing is the different effect of cilostazol and sildenafil on the disease. However, there are several major concerns must be addressed.

Major comments:

1) Author please explain the hypothesis of your study, is it only to find out the efficacy the inhibitors? As it is not clear with your experimental plan.

2) The author used a dose of 1 mg/day for each drug, how was this optimization done? Other studies often used 30-60 mg/day. The author should provide a dose optimization data as the dose author used for the study is considerably low and maybe that’s why lack of therapeutic effects observed. Pharmacokinetic data would be helpful.

3) Please justify why author chose 84 days of administration of treatments?

4) The BAPN concentration used in this study (0.5% wt/vol) which showed high mortality rates. There are previous study with low concentration (0.1-0.3%) as we know the BAPN effects are highly dose dependent. Author needs to justify dose choice based on their hypothesis or may be it is related to model also.

5) Why does the author only provide one time point mass spectrometry data? We know that the half-lives of cilostazol and sildenafil in plasma mice are relatively short (cilostazol: 2-4 hours, sildenafil: 0.3-2 hours).

6) Author did mass spectrometry which confirm the drug presence in plasma but did not address wheather the concentration of drugs were maintained throughout the experimental period or not as the half-lives of both drugs are relatively short

7) The author did bulk RNA sequencing, I don’t know how it will be helpful to author rather single cell sequencing will be helpful but I can assume the author has large data set but there is no evidence (eg. Bubble plot) mechanistic insight of drug effects or any molecular readouts in downstream pathways and author did not mentioned which cell types are involved in this mechanism. So the study lacks mechanistic investigation beyond RNA- sequencing data.

8) Author did not included any data on PDE enzyme activity , cAMP/cGMP levels or any signaling related study which limits its focus why drugs were ineffective?

9) The author states in the discussion section "These results indicate that BAPN-induced thoracic aortopathy is mediated by mechanisms distinct from other aortopathy models" by reinforcing this point with evidence or by emphasizing the ECM-dominant and non-inflammatory BAPN pathology that limits efficacy.

10) This study lacks histological evaluation, my suggestion is to include immunostaining data which would deepen the mechanistic insights.

11) The author should explain the novelty of this study in relation to other published studies as the present condition of the study only reflects the characterization of two drugs efficacy in thoracic region otherwise author should provide mechanistictic explanation for their negative findings.

Minor Comments:

1) In the abstract section line 29 author started with the describing about the disease but why “Phosphodiesterases (PDEs) regulate intracellular cyclic nucleotide concentrations” suddenly came please explain.

2) In the Introduction section line 46 “A key pathological feature of thoracic aortopathy and changes” Please check the grammer.

3) In line 53 author mentioned “This mouse model” which mouse model author wants to mention, please include that.

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Reviewer #3: No

**********

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Attachment

Submitted filename: Comments.docx

pone.0322434.s004.docx (16.8KB, docx)

Decision Letter 2

Jeffrey Isenberg

12 Aug 2025

β-aminopropionitrile-induced thoracic aortopathy is refractory to cilostazol and sildenafil in mice

PONE-D-25-15324R2

Dear Dr. Sawada,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Jeffrey S Isenberg, MD, MPH

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

The authors provided a second revised draft of their manuscript that addressed the comments put forward by the reviewer. Thier extra effort is appreciated.

Reviewers' comments:

Acceptance letter

Jeffrey Isenberg

PONE-D-25-15324R2

PLOS ONE

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