Modulation of Endothelial Progenitor Cell Secretome by LXW7-DS-SILY: Demographic-Specific Extracellular Vesicle Signatures for Enhanced Angiogenesis and Precision Wound Healing

Introduction: : Wound healing is a complex process involving inflammation, tissue formation, and remodeling, which can be significantly impaired in certain populations. Restoring normal blood flow and matrix formation is crucial for effectively healing full-thickness wounds. In this context, engineering the stem cell microenvironment using LXW7-DS-SILY has emerged as a promising therapeutic approach. LXW7-DS-SILY was formed by conjugating a decorin mimetic (DS-SILY) with LXW7, a cyclic peptide that targets the αvβ3 integrin receptor. Extracellular vesicles (EVs) play a critical role in cell-to-cell communication during tissue repair through their protein cargo. This study aimed to characterize how engineering the endothelial progenitor cell (EPC) microenvironment with LXW7-DS-SILY affects EV protein signatures and investigate demographic-specific differences to understand molecular disparities in wound healing outcomes between population groups. We hypothesized that LXW7-DS-SILY treatment would enhance the pro-angiogenic potential of the EV secretome and that demographic factors (race and sex) would influence these responses.

Materials and

Methods: : Two complementary approaches were employed: 1) Mass spectrometry-based proteomic characterization (LC-MS/MS) was performed on EVs isolated from four demographic groups: Caucasian males, Caucasian females, African American males, and African American females (n=3 per group); and 2) Proteomic analysis of EVs from endothelial progenitor cells (EPCs) treated with LXW7-DS-SILY (10 μM), DS-SILY (10 μM), or control for 48h. EVs were isolated using ultracentrifugation and characterized by Transmission Electron Microscopy, Flow Cytometry, and Tunable Resistive Pulse Sensing (TRPS). MaxQuant and Proteome Discover were used for label-free quantification. Bioinformatic analysis, including Gene Ontology enrichment, pathway analysis, and protein interaction network mapping, was conducted. Tube formation assays were performed to validate the functional impact of treatment-modulated EVs on angiogenesis. EVs isolated using ultracentrifugation showed spherical morphology by TEM, with average sizes of 97±33nm. Flow cytometry confirmed expression of EV-specific tetraspanins (CD9, CD63, CD81) across all groups.

Results, Conclusions, and Discussions:: Demographic analysis revealed a core proteome of 626 proteins shared across all groups, with sex emerging as a stronger differentiating factor than race. Male and female subjects showed distinct EV protein profiles, with males exhibiting higher expression of proteins involved in cytoskeletal organization and VEGFA-VEGFR2 signaling, while females demonstrated enrichment in coagulation pathways and PI3K-Akt-mTOR signaling. Among racial differences, Black males showed distinct enrichment in intermediate filament organization and aerobic glycolysis, critical for endothelial cell migration and energy supply during vessel formation. White subjects demonstrated higher expression of a gene module containing pro-angiogenic pathways related to ECM remodeling and vessel stabilization. For LXW7-DS-SILY treatment, proteomic analysis revealed significant upregulation of angiogenic proteins in EVs compared to DS-SILY and control. In the LXW7-DS-SILY vs. Control comparison, 45 proteins were upregulated and 22 downregulated, indicating that LXW7-DS-SILY enhanced the regulatory impact on the EPC secretome. Key proteins upregulated in the LXW7-DS-SILY-treated group included growth factors (IGF2, NRP1, FGFR1) and angiogenic regulators (ANGPT2, THBS1, CD59, IGFBP7). Gene Ontology analysis showed significant enrichment in blood vessel development, angiogenesis, and wound healing processes. Importantly, ongoing analysis is examining demographic-specific responses to LXW7-DS-SILY, with preliminary predictions suggesting females show stronger upregulation of estrogen-responsive angiogenic pathways (VEGF, eNOS) and ECM remodeling proteins, while males may exhibit enhanced expression of cytoskeletal reorganization proteins, metabolic enzymes, and stronger enhancement of integrins beyond αvβ3 (target of LXW7). Racial differences manifest in inflammatory regulation, oxidative stress management, and differential patterns of growth factor receptor expression. Tube formation assays confirmed that EVs from LXW7-DS-SILY-treated EPCs markedly enhanced vascular network formation compared to control and DS-SILY treatments. This study provides the first comprehensive investigation of how engineering the stem cell microenvironment with LXW7-DS-SILY modulates the EPC secretome to enhance angiogenesis, while also characterizing demographic-specific EV signatures with direct implications for wound healing. Additionally, the observed differences in angiogenic pathways between demographic groups may contribute to population-specific variations in wound repair efficacy. Future studies will include cross-demographic testing, where EVs isolated from one group will be applied to endothelial cells from other groups.

Acknowledgements and/or References (Optional):: Acknowledgements:
UC Davis Stem Cell and Gene Therapy Training Program 2.0
Georgia Institute of Technology College of Engineering President’s Postdoctoral Fellowship

Reference:
Dartora VFC, Carney R, Wang A, Qiu P, Panitch A. Extracellular matrix ligands modulate the endothelial progenitor cell secretome for enhanced angiogenesis. Acta Biomater. 2025 Mar 15;195:240-255.