P1 - Leveraging Apoptotic Neutrophils With The Application of Biomaterial for Improved Wound Healing Repair

Saturday, October 11, 2025

10:00 AM - 11:00 AM PDT

Location: Exhibit Hall F, G & H

Presenting Author(s)

Ronald Nguyen

Undergraduate Researcher
University of California, Irvine
Huntington Beach, California, United States

Co-Author(s)

LD

Limor David

Lab manager
UC Irvine, United States

Primary Investigator(s)

Wendy F. Liu, PhD

Professor
University of California, Irvine
Irvine, California, United States

Introduction: : Efferocytosis, the clearance of apoptotic cells by phagocytosis, plays a vital role in resolving inflammation and initiating tissue regeneration. During this process, macrophages (Mɸ) transition from a pro-inflammatory to an anti-inflammatory phenotype, promoting the secretion of regenerative factors such as vascular endothelial growth factor (VEGF), transforming growth factor β (TGFβ), and fibroblast growth factor (FGF). These molecules facilitate angiogenesis, matrix remodeling, and cellular recruitment. At a transcriptional level, Mɸ exposed to apoptotic neutrophils (ANs) exhibit elevated expression of VEGFA, PDGFB, FGF2, IGF1, and HGF, which are genes essential for endothelial proliferation, fibroblast migration, and epithelial regeneration. Preliminary in vivo studies suggest that delivering ANs within a GelMA hydrogel scaffold enhances angiogenesis, underscoring the need to isolate apoptotic components responsible for macrophage-mediated tissue repair.

Materials and

Methods: : C57BL/6J mice were used to harvest macrophages and neutrophils from bone marrow. Macrophages are cultured in macrophage colony-stimulating factor (MCSF) containing medium and differentiated for a week prior, while neutrophils are separated from the lysed pellet and filtered through a magnetic column using neutrophil-negative magnetic beads. These neutrophils are subjected to five conditions: (1) spontaneous apoptosis, (2) 42°C water bath, (3) ultraviolet radiation, (4) staurosporine (STS), and (5) midostaurin (Mido) treatment to analyze distinct apoptotic populations. While the ladder conditions face their respective treatments, spontaneous apoptosis is treated with incubation in 1% FBS medium for 24 hours. The cells and supernatant are collected and applied to macrophages in a ratio of 5:1 for 24 hours. These cells and supernatants are then collected to analyze for flow cytometry, ELISA (TGFβ & VEGF), qPCR (VEGFA, PDGFB, TGFB1, FGF2, IGF1, HGF), and western blot to evaluate macrophage responses and neutrophil populations.

Results, Conclusions, and Discussions:: Validation of the AN populations confirmed that all treatment conditions effectively induced apoptosis, as indicated by significant shifts in early and/or late apoptotic population profiles (Fig.1A). Among these, STS and Mido treatments generated the highest proportions of early apoptotic cells, in contrast to UV and 42°C heat treatment, which showed a dominance in late stage apoptosis, When applied to Mɸ, STS- and Mido-treated ANs elicited the strongest pro-angiogenic responses inducing the highest VEGF secretion with STS in particular showing significant elevated expressions in VEGFA, FGF2, and HGF gene expressions (Fig.1B-C). These findings suggest that STS and Mido treatments generate ANs capable of enhancing Mɸ-mediated angiogenesis, primarily through upregulation of key regenerative factors. The increased expression of HGF and FGF2 further implies the role of Mɸ in the migration of hepatocytes, epithelial cells, and endothelial cells to the wound site. Importantly, the predominance of early apoptotic groups is consistent with prior findings supporting anti-inflammatory Mɸ phenotypes. In contrast, progression to secondary necrotic stages (late apoptosis), as seen with a compromised membrane, can release DAMPs and immunogenic nucleoprotein complexes that may trigger mixed or inflammatory responses depending on the microenvironment [1] [2].

This study demonstrates that ANs, particularly those treated with staurosporine and midostaurin, effectively reprogram Mɸ toward an angiogenic and anti-inflammatory phenotype. These findings position ANs as potent bioactive modulators of the wound healing process and support their potential incorporation into regenerative therapeutic strategies. Moreover, the observed differences in macrophage responses to immune versus stromal apoptotic bodies highlight the specificity of efferocytic signaling pathways and their dependence on cell lineage. Given the limited availability of T-cells in healthy individuals and their potential to trigger autoimmune responses, ANs represent a more practical and immunologically compatible candidate for targeted immunomodulation in tissue repair applications.

Acknowledgements and/or References (Optional): : [1] V. A. Patel et al., “Apoptotic Cells, at All Stages of the Death Process, Trigger Characteristic Signaling Events That Are Divergent from and Dominant over Those Triggered by Necrotic Cells,” Journal of Biological Chemistry, vol. 281, no. 8, pp. 4663–4670, Feb. 2006, doi: https://doi.org/10.1074/jbc.m508342200.
[2] M. Sachet, Y. Y. Liang, and R. Oehler, “The immune response to secondary necrotic cells,” Apoptosis, vol. 22, no. 10, pp. 1189–1204, 2017, doi: https://doi.org/10.1007/s10495-017-1413-z.

This work was supported by the Cardiovascular Innovation and Research Center (CIRC) and the University of California, Irvine, whose resources and collaborative environment were instrumental in facilitating this research. I would like to sincerely thank the mentors and researchers at the Liu Lab for their invaluable guidance, technical expertise, and continuous support throughout the development of the project.