Abstract
Background
Urinary extracellular vesicles (EVs) have gained increasing interest in recent years as a potential source of noninvasive biomarkers of diseases related to urinary organs, but knowledge of the mechanism is still limited. The current study sought to clarify the mechanism of urinary EVs behind di-(2-ethylhexyl) phthalate (DEHP)-induced hypospadias via PFN2 delivery.
Method
PFN2 expression in hypospadias was predicted by bioinformatics analysis. Following the induction of a hypospadias rat model using DEHP, rats were injected with EVs and/or underwent alteration of PFN2 and TGF-β1 to assess their effects in vivo. The extracted rat urothelial cells (UECs) were co-cultured with EVs extracted from urine for in vitro experiments.
Result
Microarray analysis predicted poor PFN2 expression in hypospadias. Upregulated PFN2 was found in urinary EVs, and restrained epithelial-mesenchymal transition (EMT) was observed in DEHP-exposed rats. Urinary EVs or PFN2 overexpression increased SMAD2, SMAD3, and TGF-β1 protein expression and SMAD2 and SMAD3 phosphorylation in UECs and DEHP-exposed rats. UEC migration, invasion, and EMT were augmented by EV co-culture or upregulation of PFN2. Of note, the silencing of TGF-β1 counterweighed the effect of PFN2. Besides, EV co-culture or overexpression of PFN2 or TGF-β1 elevated the body weight, anal–genital distance (AGD), anal–genital index (AGI), and EMT of DEHP-exposed rats.
Conclusion
In summary, urinary EVs activated the SMAD/TGF-β1 pathway to induce EMT via PFN2 delivery, thus protecting against DEHP-induced hypospadias.
Graphical Abstract
(1) EMT in epithelial cells inhibits DEHP-induced hypospadias.
(2) Urine-derived EVs deliver PFN2 to promote EMT in epithelial cells.
(3) PFN2 can activate the SMAD/TGF-β1 signaling axis.
(4) Urine-derived EVs can transmit PFN2 to activate the SMAD/TGF-β1 signaling axis, thus promoting EMT and inhibiting the occurrence of hypospadias.
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Data Availability
The data and materials of the study can be obtained from the corresponding author upon request.
Code availability
Not applicable.
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Funding
This study was funded by the Science and Technology Projects in Guangzhou (202102020097, 202201020598) and the Guangzhou Institute of Pediatrics/Guangzhou Women and Children’s Medical Center (pre‐NSFC‐2018‐016, YIP‐2018‐021, GWCMC2020‐4‐009) and the Natural Science Foundation of Guangdong Province, China (2019A1515011178).
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Shibo Zhu and **angliang Tang contributed to the conception and design of the study; ** Zhang and Dian Li contributed to the acquisition of data; **hua Hu contributed to the analysis and interpretation of data; Shibo Zhu and **aofeng Gao contributed to drafting the article; **angliang Tang and Wei Jia contributed to revising the article critically for important intellectual content; all of the authors approved the final version to be submitted.
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This study was approved by Guangzhou Women and Children’s Medical Center, Guangzhou Medical University and in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health. Adequate measures were taken to minimize suffering of the included animals. Consent to participate is not applicable.
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Zhu, S., Tang, X., Zhang, J. et al. Urinary extracellular vesicles prevent di-(2-ethylhexyl) phthalate-induced hypospadias by facilitating epithelial–mesenchymal transition via PFN2 delivery. Cell Biol Toxicol 39, 2569–2586 (2023). https://doi.org/10.1007/s10565-023-09838-1
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DOI: https://doi.org/10.1007/s10565-023-09838-1