Modulation of the secretome profile of endothelial progenitor cells with bioactive glycosaminoglycan materials to improve wound healing

Introduction: Wound healing involves inflammation, tissue formation, and remodeling to repair damage. However, this process can be impaired, leading to chronic wounds. In developed nations, 1 to 2 percent of individuals will experience a chronic wound in their lifetime. In the United States alone, chronic wounds affect approximately 6.5 million patients, often resulting in persistent pain, reduced mobility, prolonged hospitalization, financial strain, and higher rates of morbidity and mortality. The economic burden is significant, with over $25 billion spent annually on treating wound-related complications. Restoring normal blood flow and matrix formation is crucial for effectively healing full-thickness wounds. In this context, LXW7-DS-SILY has emerged as a promising therapeutic approach. It was formed by conjugating a decorin mimetic (DS-SILY) with LXW7, a cyclic peptide that targets the αvβ3 integrin receptor. LXW7-DS-SILY has shown promise in enhancing wound strength, regulating collagen fibril formation, and activating VEGFR2 on endothelial progenitor cells (EPCs) to promote cell proliferation, migration, and angiogenesis. EPCs play a crucial role in angiogenesis, and their secretome contains a rich composition of proteins and extracellular vesicles (EVs) that are critical for cell signaling and tissue regeneration. Understanding the modulation of the EPC protein secretome profile may hold key therapeutic potential for enhancing angiogenesis and wound healing. This study aimed to investigate whether LXW7-DS-SILY modulates the EPC secretome profile, particularly focusing on the protein content. We hypothesized that LXW7-DS-SILY would enhance the pro-angiogenic and regenerative potential of the EPC secretome by upregulating proteins involved in angiogenesis, tissue remodeling, and wound healing.

Materials and

Methods: A proteomic characterization of the secretome was performed after EPC treatment with LXW7-DS-SILY (10 M) or DS-SILY (10 M) for 48 h and compared to untreated (control). Extracellular vesicles (EVs) were isolated by ultracentrifugation and characterized with Transmission Electron Microscopy (TEM), Flow Cytometry for surface proteins, and Nanoparticle Tracking Analysis (NTA) for size and concentration. Mass spectrometry (LC/MS-MS) was performed to analyze the secretome's EV protein composition and soluble content. The protein identification was performed using the Spectronaut® software to estimate protein fold changes among conditions. The analysis of proteins for overrepresented Gene Ontology (GO) biological processes was conducted using Panther, employing a Fisher’s Exact test with FDR correction for multiple tests and DAVID 6.8. Selective proteins were inputted into the String database to visualize protein interactions and analyzed with Cytoscape 3.10.2. To gain a deeper insight into how alterations in the EPC secretome impact angiogenesis, a tube formation assay was conducted on the entire secretome as well as its EVs and soluble components individually. Sample Progression Discovery (SPD) was employed to correlate proteomic data with angiogenesis to integrate multi-omics data. This approach aimed to identify potential groups of proteins responsible for enhancing angiogenesis and determine whether they were associated with extracellular vesicles or the soluble fraction of the secretome.

Results, Conclusions, and Discussions: The analysis revealed significant changes in protein expression between treatments. In the DS vs. Control comparison, 17 proteins were upregulated, and 9 were downregulated. In the LXW7-DS-SILY vs. Control comparison, 45 proteins were upregulated, and 22 were downregulated, indicating that LXW7-DS-SILY significantly enhanced the regulatory impact on the EPC secretome. Treatment with LXW7-DS-SILY significantly improved tube formation compared to DS-SILY or Control. SPD analysis and module clustering highlighted distinct patterns between treatment groups. A module of 87 genes was significantly associated with the LXW7-DS-SILY-treated samples compared to the DS-SILY and Control groups. Key proteins upregulated in the LXW7-DS-SILY-treated group included growth factors (IGF2, NRP1, FGFR1) and angiogenic regulators (ANGPT2, THBS1, CD59, IGFBP7). These proteins promote angiogenesis, collagen fibril formation, and EPC proliferation. Further analysis revealed that EVs were primarily responsible for the majority of the angiogenic activity, confirmed by an angiogenesis assay using EVs from EPCs treated with LXW7-DS-SILY, DS-SILY, and controls. GO enrichment analysis was performed on the EVs to further understand the biological processes influenced by LXW7-DS-SILY. The analysis revealed significant enrichment in blood vessel development, angiogenesis, and wound healing processes. Positive regulation of angiogenesis and blood vessel endothelial cell migration were also significantly enriched, with fold enrichments of 10.29 and 15.53, respectively. A protein interaction network analysis using STRING and Cytoscape highlighted intricate interactions between key proteins influenced by LXW7-DS-SILY treatment. The network revealed central nodes involving FN1, THBS1, and MMP2, known to play pivotal roles in angiogenesis and wound healing. Other notable proteins included IGF2, FGF2, FGFR1, and ANGPT2, all of which contribute to endothelial cell proliferation, migration, and extracellular matrix remodeling.
In conclusion, LXW7-DS-SILY significantly modulates the EPC secretome to enhance angiogenic potential, mainly through upregulating angiogenesis-related proteins and pathways. The analysis revealed that EVs from the EPC secretome were particularly enriched in pro-angiogenic proteins, contributing to blood vessel development, angiogenesis, and wound healing processes. LXW7-DS-SILY treatment significantly improved tube formation compared to DS-SILY and the control, with EVs playing a pivotal role in this enhancement. These results support LXW7-DS-SILY as a promising therapeutic agent for improving wound healing by modulating the EPC secretome profile.

Acknowledgements (Optional):