Ex vivo minoxidil treatment increases elastic fiber production in the murine vaginal wall

Introduction:: Elastic fibers play an important role in maintaining the structure and function of soft tissues throughout the body including the skin [1], cardiovascular system [2], and pelvic organs [3]. Disruption of elastic fiber microstructural organization – through different processes including aging, injury, and disease – is detrimental to elastic fiber homeostasis and alters soft tissue mechanical properties. In particular, pathological conditions of the female pelvic floor are associated with compromised elastic fiber integrity. Pelvic organ prolapse (POP) is defined as a gradual downward shift of the uterus, cervix, and bladder to an extent that results in the bulging of the descendent organs to or through the vaginal canal. Biochemical examination of vaginal samples from women with POP shows that elastin protein levels are negatively correlated with POP severity [4]. As there are currently no pharmacological approaches to manage POP symptoms, one-in-eight women affected by POP will ultimately require surgical intervention to restore the anatomical structure [5]. Motivated by prior work investigating pharmacological approaches to induce elastic fiber formation in the cardiovascular system [6, 7], in the current study we investigated the ability of the anti-hypertensive drug minoxidil to promote elastogenesis in ex vivo vaginal tissue explants. Minoxidil, and its active metabolite minoxidil sulfate, is a potent vasodilator that activates ATP-sensitive potassium channels and is known to lower blood pressure and induce hair growth [6]. To date, the potential for minoxidil to induce elastogenesis has not been explored in tissues outside the cardiovascular system.

Materials and Methods:: Adult wild-type female mice were anesthetized using isoflurane prior to euthanasia and removal of the intact pelvic organs including the vagina, cervix, and uterus. Following tissue isolation, the reproductive organs were randomly separated into two treatment groups and placed in DMEM culture media containing 1% antibiotic-antimycotic solution and 10% fetal bovine serum (FBS) with or without 0.24 mM minoxidil sulfate. Tissue explants were cultured at 37°C and 5% CO2 for 7 days and the media was replaced every 24 hours. To assess changes in elastic fiber synthesis induced by minoxidil treatment, we investigated changes in elastin expression at the mRNA and protein level; tropoelastin gene expression (Eln) was measured by qPCR and soluble α-elastin protein was measured by ELISA. To further visualize changes in the elastic fiber architecture of the vaginal wall following minoxidil treatment, cultured vaginal tissue explants were embedded, cryosectioned, fixed, and permeabilized prior to immunostaining for elastin (in red) and alpha-smooth muscle actin (αSMA; in yellow). Immunofluorescent images were collected at the same imaging settings (exposure time, etc.) to allow for semi-quantitative comparisons across treatments.

Results, Conclusions, and Discussions:: Results: Comparison of tropoelastin gene expression (Eln) and elastin protein levels in vaginal tissue explants revealed that minoxidil treatment significantly increased both gene (p < 0.05; Fig. 1A) and protein levels (p < 0.01; Fig. 1B) of elastin after 7 days of ex vivo culture, relative to control. Spatially-resolved analysis of immunofluorescence images from control and minoxidil-treated tissue revealed an increase in elastin staining intensity in the subepithelium (p < 0.05; Fig. 1C) and a trend toward increased elastin staining intensity in the muscularis. Minoxidil treatment also increased the staining intensity of the contractile protein αSMA in the muscularis layer of the vaginal wall, albeit not statistically significant (p = 0.07; Fig. 1C).

Discussion: In the current study, we demonstrated that minoxidil treatment has the potential to alter elastin fiber formation in adult wild-type vaginal tissue explants cultured ex vivo. Our findings suggest minoxidil treatment causes increases in elastin expression not only at the gene level but also at the protein level. Although the ability of minoxidil to promote elastic fiber production in vivo requires further evaluation, our findings indicate this type of treatment strategy could effectively target the underlying biology that contributes to POP progression, which is an avenue that is currently missing in the management of POP.

Conclusion: Ex vivo minoxidil treatment of vaginal tissue has illustrated for the first time that minoxidil sulfate has the potential to increase elastic fiber synthesis in the vaginal wall. This is the first evidence of minoxidil-induced elastin alterations in tissues outside of the cardiovascular system and indicates a promising direction for future study in conditions involving pathological remodeling of the female pelvic floor. The potential for drug-induced elastogenesis in pelvic organ tissues can begin to introduce non-invasive approaches to manage the symptoms of POP.


Acknowledgements (Optional): : We acknowledge internal funds from Washington University in St Louis and the National Institutes of Health (R00 HL146951) to MRB.

References (Optional): : [1] Baumann et al., Aesthet Surg J. 2021.
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