ABSTRACT: Venous leg ulcers (VLUs) are the most common cause of leg ulcers, yet only 44% heal with standard of care, highlighting the critical need for better understanding of their cellular pathology. We used a combination of single-cell RNA-Sequencing (scRNA-Seq) and bulk RNA-Seq to identify molecular mechanisms and cellular functions contributing to VLU pathophysiology. scRNA-Seq of chronic VLUs revealed impairments in immune, lymph-endothelial, and endothelial cells, along with the underlying signaling pathways. Next, RNA-Seq was performed to asses clinical outcomes in patients receiving standard of care, with wound healing rate monitored for at least 4 weeks. Transcriptomes of healing and non-healing VLUs were compared to those of human acute wounds (AW), revealing a robust suppression of inflammatory response, lymphangiogenesis and angiogenesis in non-healing VLUs. In contrast, healing VLUs resembled gene expression signature of physiological, acute wound healing. Reduced inflammatory response resulting from impaired leukocyte transmigration and egression, with suppression of kinases’ activity, including AKT, ERK, JNK1/2, SRC, and PDGFR, underlines the non-healing VLU signature. Bioinformatic analyses pinpointed PTEN as a master regulator of these processes and signaling pathways. Indeed, increased PTEN was confirmed in non-healing compared to healing VLUs, both on scRNA-Seq and protein levels. Functional in vivo murine studies confirmed that pharmacological PTEN inhibition enhanced immune response and proinflammatory signals, angiogenesis and lymphangiogenesis which resulted in accelerated wound closure. We identified PTEN as a master regulator of non-healing phenotype controlling multiple key processes responsible for impaired healing, underscoring its potential use as diagnostic and therapeutic target to promote wound closure in VLUs.
