SP44. High Throughput In Vitro Platform For Mechanistic Exploration Of Androgen Effects On Wound Angiogenesis And Repair
Year: 2024
Type: article
Abstract: Purpose: Our group has shown greater hypertrophic scarring in transgender males receiving testosterone (T) therapy. Similar effects are observed in rodent and pig WH models, proceeding via pro-inflammatory wound effects of testosterone. A central paradox is that a pro-inflammatory T effect is observed in WH, yet systemically, T generally exerts anti-inflammatory physiologic effects. Further, in vitro models of WH cells show anti-inflammatory reactions to T therapy compared to animal models. The lack of an in vitro model has stymied progress in understanding T-WH mechanisms. Testosterone has been shown to promote angiogenesis in various contexts, such as after myocardial infarction. We hypothesized that T treatment induces angiogenic wound effects, known to induce inflammation via the VEGF-TNFa axis, and the absence of the angiogenic capacity is the key deficiency in past in vitro T-WH assays. Methods: Male and female human dermal fibroblasts (HDF) and human umbilical vein endothelial cells (HUVEC) were grown and maintained in FGM-2 and EGM-2 culture media respectively under standard conditions. 10,000 HDF and 2,000 HUVEC were seeded into each well. R1881, a testosterone analogue with greater solubility and no capacity for aromatization to estradiol, was used for androgen receptor stimulation. There were 9 media treatment conditions, based on media spiked with 0, 10, and 50nM R1881 and 0, 10, and 40 ng/mL vascular endothelial growth factor (VEGF). Brightfield images were collected every day. The supernatant was collected and the media was replaced every two days. Plates were harvested on days 7 and 10. Immunofluorescent imaging and Z-stacking were performed on Zeiss AxioObserver after staining with DAPI, CD31, CD144, Vimentin, and FAP antibodies and cell tracker dyes. Area fraction and branching were analyzed using ImageJ. Total protein and VEGF concentration analyses were performed through BCA and ELISA assays. Results: Increased angiogenesis was observed with increasing levels of VEGF stimulation. Exposure to R1881 increased numbers of branching (p<0.001) and triple-points (p=0.03) compared to non R1881-treated wells. Supraphysiologic dosing of R1881 led to a slight decrease in response. Conclusion: We developed a novel in vitro co-culture that simulates the wound cell milieu to study how testosterone affects angiogenesis and inflammation. Using 3D fluorescent microscopy and angiogenesis measures, high-throughput analysis of experimental permutations is feasible. We found that androgen receptor activation induces pro-angiogenic stimulus in a fibroblast-endothelial co-culture, which is linked to pro-inflammatory effects via the angiogenesis-inflammation paradigm. However, the presence of VEGF is also required to initiate these effects. Prior in vitro studies may have failed to recapitulate T-induced effects on WH due to lack of EC and VEGF, both of which are present in the wound environment. Studies to introduce macrophages for triple co-culture and characterize T effects on macrophage phenotype are currently under way. Future investigations will use this model to leverage experimental manipulations feasible only in vitro to identify core T-WH mechanisms, followed by in vivo validation.
Institutions: Johns Hopkins University, Brigham and Women's Hospital
Language: English
Topic: Wound Healing and Treatments
Subfield: Rehabilitation
Field: Medicine
Domain: Health Sciences
Open Access status: gold
Estimated APC paid: $2,195
OpenAlex ID: W4394953638