(WHS-P1.05) LCM-DIRECTED PROFILING OF ACUTE AND CHRONIC WOUNDS IDENTIFIES PROTEOMIC AND LIPIDOMIC SIGNATURES OF HEALING AND NON-HEALING WOUNDS
Friday, May 17, 2024
10:30 AM – 11:30 AM East Coast USA Time
Purpose: The purpose of our study was to utilize unbiased proteomic and lipidomic approaches to identify potential biomarkers of healing and non-healing wounds.
Methods: For acute wounds, we topically treated ex vivo human skin with deuterium oxide (D2O), whose incorporation into newly synthesized macromolecules was assessed at 24, 48, 72 and 96hr post treatment (n=4). Similarly, for chronic wounds we utilized diabetic foot ulcer (DFU) and venous leg ulcer (VLU) specimens (n=3). Harvested tissues were cryosectioned & freeze dried, with areas of interest dissected using LCM microscopy and sequential sections subjected to either lipidomic or proteomic analysis using time of flight secondary ion mass spectrometry (ToF-SIMS) or trapped ion mobility spectrometry (timsToF-Ultra) with a high-spatial resolution LMIG analytical beam (respectfully). Mass-to-charge ratio (m/z) was utilized to identify various lipid and peptide species using Spectronaut 18, thus allowing for hierarchical clustering of samples, followed by STRING-DB and DAVID analysis for functional annotation of identified clusters of different lipidomic and proteomic species.
Results: TOF-SIMS detected fatty acids 16:0 and 16:1 (255.2, 253.2 m/z), 18:0, 18:1 and 18:2 (283.3, 281.2, 279.2 m/z), and cholesterol sulfate (465.3 m/z) in both acute and chronic wounds with the latter exhibiting close to 200-fold increase in epidermis of both DFUs and VLUs. Conversely, long chain fatty acids (24:0, 25:0, 26:0 and 28:0), as well as ceramides were almost devoid from the healing epidermis and were primarily localized to granular and cornified layers away from the healing epidermis in acute wounds and throughout the entirety of both DFUs and VLUs. On the other hand, proteomic analyses identified a number of proteins that exhibited differential expression in either healing epidermis or dermis in comparison to their counterparts away from the wound (including but not limited to: ALDOC, ANXA3, ATPA, BAF, CALD1, CALM1, CAV1, DESP, EPIPL, FAK2, G3PT, GELS, H11, K1C24, KRT36, K2C6A, PDIA1, POSTN, RL12, ROA1, SPB5, VINC) with a relative difference in abundance >2.0 fold. Moreover, we were able to identify a number of differentially expressed peptides between acute and chronic wounds, within both epidermal and dermal compartments (A1AG, CALM2, CCN1, DCD, FIBB, FNDC1, H2A1J, HBB, K1C9, K22E, MACF1, OR2T2, O2T35, PGS2, RLA2, S10A9, VIME, among others).
Conclusions: Together our data identify a novel method for detection of proteomic and lipidomic changes in acute and chronic wounds. Moreover, identified signatures serve as targets for development of novel topical formulations for accelerated wound closure, which we are actively pursuing.