Project Summary This project proposes to use wound fluid analyses captured from discarded wound dressings to develop biomarkers that can predict healing outcome and monitor healing progression of Venous Leg Ulcers (VLUs). VLUs represent a specific case of chronic ulcers occurring in the gaiter area in the setting of chronic venous insufficiency and account for about 70% of all chronic leg ulcers. Considering the aging of the population a concerning picture emerges: more ulcers occurring in sicker patients which are ever more expensive to treat. Despite recent advances, more than 50% of patients with VLUs fail to heal with standard care. One of the major obstacles to improving outcomes is the inability to predict early on who will and who will not respond to standard of care. Therefore, there is an urgent and unmet need to predict healing outcomes and to differentiate healing from non-healing VLUs. Due to dynamic molecular changes within the wound, its microenvironment can be tested and monitored, which offers an opportunity to obtain key information regarding the status of wound biology. Moreover, quantification of such molecular changes that reflect wound healing status would provide important tools that can guide treatment approach, directly impacting the clinical outcomes. Thus, both predictive and monitoring biomarkers are needed to enable prospective tailoring of therapies to efficiently: 1) maximize the treatment outcomes, 2) target more aggressive treatment to only those patients who need it and 3) develop personalized approaches to each VLU. In the initial discovery phase, we will enroll 30 VLU patients (10 healers and 20 non-healers) to identify a set of protein biomarkers that predict healing outcomes (Aim 1); those that can serve as monitoring biomarkers (Aim 2). We will extract wound fluid from discarded wound dressings and quantify proteins by using mass spectrometry (LC-MS/MS) and correlate them to healing outcomes at week 4. We have generated preliminary data that support feasibility of the overall approach and confirm that wound dressings (either analyzed in real time or stored frozen) of any type can be utilized for biomarker quantification. We have also identified spatially distinct localization of molecules at the center vs perimeter of the wound dressing, resulting in specific profiles that reflect the biology of the wound bed and wound edge respectively. This project will provide a unique opportunity to capitalize on easy access of discarded wound dressings as a source of wound biomaterial (exudate) that can serve for biomarker detection. It will not only identify and validate specific set of biomarkers that can predict healing outcome and/or monitor healing progression but will also provide streamlined method of detection that can be readily implemented.