Persistent PTEN signaling underlies the non-healing phenotype in venous leg ulcers

Summary: Abstract Body: Venous leg ulcers (VLUs) are common chronic wounds with only 44% healing rate with standard of care. This highlights the need for better understanding of their molecular and cellular pathology to improve treatment. In this study, we investigated the mechanisms that influence the clinical progression of VLU healing. We performed single-cell and bulk RNA sequencing on VLU samples and compared them with human acute wounds (AW) using Ingenuity Pathway Analysis. Validation was done through immunostaining, qPCR, flow cytometry, and proteome arrays. Additionally, a murine wound model was used for pharmacological confirmation of findings. Our single-cell analysis showed impaired immune and lymph-endothelial functions in VLUs. Comparison of healing versus non-healing VLUs revealed that healing VLUs exhibited immune responses similar to acute wounds, while non-healing VLUs had suppressed inflammation and lymph-angiogenesis. This inadequate inflammatory response in non-healing VLUs was linked to impaired cell processes, including reduced transmigration, egression and chemotaxis, and decreased immune recruitment. Furthermore, signaling pathways involving AKT, ERK, SRC, STAT, and PDGFR were suppressed in non-healing ulcers. Upstream regulator analysis identified PTEN as a critical negative regulator of these pathways. Consistently, PTEN protein expression was significantly higher in non-healing VLUs, indicating its central role in driving the non-healing phenotype. PTEN inhibition in murine wound model accelerated wound closure, enhanced granulation tissue formation, and promoted inflammatory response. Our findings position PTEN as a critical regulator of non-healing VLUs, offering potential targets for new therapeutic interventions to improve healing outcomes. Jelena Marjanovic², Beatriz Abdo Abujamra², Natasa Strbo¹, Anthony J. Griswold³, Ivan Jozic², Raji R. Nagalla², Rivka Stone², Hadar Lev-Tov², Robert S. Kirsner², Irena Pastar², Marjana Tomic-Canic² 1. Department of Microbiology and Immunology, University of Miami Miller School of Medicine, Miami, FL, United States. 2. Department of Dermatology, University of Miami Miller School of Medicine, Miami, FL, United States. 3. John P. Hussman Institute for Human Genomics, University of Miami Miller School of Medicine, Miami, FL, United States. Stem Cell Biology, Tissue Regeneration and Wound Healing