Introduction: Lichen Sclerosus (LS) is an underdiagnosed inflammatory skin condition that primarily affects the skin in the genital and anal regions. Although LS can affect anyone, it is predominantly diagnosed in postmenopausal women and, to a lesser extent, men, prepubertal children, and adolescents. In women, LS manifests as chronic whitish atrophic patches in the vulvar region called vulvar lichen sclerosus (VLS), causing significant discomfort and lowering the quality of life. There has been no comprehensive research on VLS to date, and the overall mechanism of this disease is still mostly unknown. The primary indicator of lichen disease is pruritus, or itching, which the patient or healthcare practitioner may ignore or mistake for a yeast infection, irritation, or scarring dermatitis. The most common hypothesis is that abnormal wound healing causes a build-up of extracellular matrix (ECM) material known as sclerotic matter, which decreases tissue pliability and ultimately causes function loss. Although steroids often prevent disease progression, they do not reverse changes that have already occurred, and in some patients, they fail to prevent progression. In VLS, scarring of vulvar structures may lead to cancer, although the link between scarring and cancer is unclear. VLS is the only LS condition associated with an elevated risk of cancer, so there is a critical need to understand the mechanisms driving this disease process.
Materials and
Methods: Tissue samples were collected from scarred and visibly unaffected areas of 8 VLS patients to create fibroblast strains for study. We developed an in vitro wound healing model using a scratch assay on confluent monolayers of cells. To visualize nuclei and cell bodies, cells were stained with Hoechst and CellTracker Red fluorescent dyes. The cells were treated with either vehicle control or TGF-β. Our study focused on several genes of interest. ACTA2, which encodes α-smooth muscle actin (αSMA), was selected as a marker of myofibroblast production and EN1 (Engrailed-1) was chosen as a potential marker for scarring. To assess cellular responses to injury, we examined Ki67, p53, and BAX to determine whether cells were proliferating or undergoing apoptosis. Quantitative PCR was used to analyze gene expression, with 18S rRNA serving as our normalization control. Additionally, we performed an Alamar Blue assay to measure cell viability. To create a more accurate model of VLS conditions, we are developing a Collagen assay. This approach employs varying collagen consistencies to mimic the properties of scar tissue. We can simulate mechanical stress by applying friction and abrasion using various rough materials, aiming to replicate the stresses experienced in VLS more realistically.
Results, Conclusions, and Discussions: ACTA2 and EN1, both associated with scarring, showed reduced expression in scratched VLS cells compared to non-scratched controls when our cells were stained for imaging. This was contrary to our initial hypothesis that these markers would increase during the wound healing process. ACTA2 decreased significantly (p < 0.05), while EN1 showed a similar but not statistically significant downward trend. However, we repeated this experiment without staining the cells for visualization and no longer saw a difference in ACTA2 and EN1. We have subsequently determined that the staining protocol may affect cellular viability and growth. Interestingly, p53, a cell cycle marker involved in DNA repair and prevention of unchecked cellular proliferation showed a significant increase in scratched as opposed to non-scratched cells, regardless of staining. BAX, a marker of cell death remained unchanged, while Ki67, a marker of cell proliferation trended towards an decrease in scratched areas. Scratching induces cellular damage and may reduce proliferation, but that is currently unclear from our data. Scratched wells exhibited higher fluorescence in the Almar Blue assays, which suggests that there are a greater number of viable cells. This could be the result of enhanced proliferation to fill the gap or a decrease in the viability of non-scratched cells. These results suggest that scratch injuries might cause cells to proliferate, focusing on quick wound closure rather than producing extracellular matrix materials. While our current scratch test model provided valuable information, it does not replicate the environment present in VLS lesions. Due to these limitations, we are now working on a more advanced Collagen-based model. This new approach aims to better mimic the properties of scar tissue and the physical stresses experienced in VLS. We hope this improved model will help us understand how different types of physical stress affect cell behavior and gene activity in VLS.
Acknowledgements (Optional): I would like to thank my mentor Megan Falsetta and my lab members Ashley Updike and Zahra Mahamed for their assistance and encouragement. I would also like to thank Maeystone Good Grant for the funding and support.
