PROJECT ABSTRACT Wounds have a sizable impact on public health: 1 out of 50 Americans has a chronic wound at any given time and the cost of wound care exceeds $100 billion per year. Similarly, hypertrophic scars with excessive scar tissue greatly impair quality of life for millions of patients. However, therapeutic solutions for wound healing (WH) remain quite limited. Sex has received growing interest as a factor in wound healing, and some studies show higher rates of non- healing wounds in men. It has long been known that in male rodents testosterone (T) impairs WH via androgen receptor effects. Sex hormone (SH)-induced changes in wound repair have been studied as a possible risk factor, but their impact is uncertain due to factors like illness that alter hormone levels. A novel and promising way to better understand this link is to examine WH in transgender patients on exogenous hormones. However, research into the effects of gender-affirming hormones has not kept pace with rapid clinical population growth. We developed new models and showed that T limits wound healing in large animals with different effects in XX vs. XY animals. Further, we have shown that patients on T have impaired wound healing. A critical knowledge gap that remains unaddressed is the set of molecular mechanisms and pathways testosterone acts through to change the wound healing response. We have identified new immunomodulatory effects of T that may mediate these effects. Conversely, estradiol (E2) may accelerate wound repair rates. Essential aspects of how E2 improves wound healing – how E2 levels affect the response, what cell subtypes E2 acts through, and whether E2 effects differ between XX and XY subjects – remain similarly unknown. We will address these gaps in understanding sex hormone modulation of WH with a program spanning three project directions, including overcoming the failure of current in vitro models to recapitulate the in vivo wound cell behavior changes induced by sex hormones (Project 1: In Vitro System), defining how testosterone modulates the evolution of the immune response during wound healing to impair closure and how this induces a fibrotic or regenerative repair response (Project 2: Mechanisms of T Immunomodulation). Finally, we will expand our work to deeply profile the effects of estradiol on immune cell subtypes and the wound cytokine milieu and define how sex chromosome complement alters this response (Project 3: Estradiol Wound Healing Mechanisms). This project will deploy innovative tools to probe SH-WH mechanisms, expanding scientific knowledge of how T and estradiol interact with sex chromosomes to regulate wound healing biology. This study seeks to transform current concepts of how hormones modulate wound repair with the dual aim of improving care for a marginalized patient population and developing potential new approaches for millions of patients with chronic wounds.

PROJECT NARRATIVE Wound healing is a major public health problem affecting millions of patients, and preliminary clinical and large animal data suggest that testosterone may impair wound repair while estrogen improves it, yet the mechanisms of these effects are poorly understood. In this project we will define the biology of how testosterone and estradiol act to change the wound immune environment and modulate the process of wound healing. This work will increase our understanding of biology in an understudied health disparity population and contribute new, fundamental knowledge of the sex hormone-tissue repair axis.