Neonatal Cardiac Extracellular Matrix as a Biomaterial for Immunomodulation

Introduction: : Neonatal mammals display a large reparative capacity in the heart not found in adults, a characteristic which drops significantly shortly after birth and then continues to decrease with age [1]. This decrease in reparative capacity is associated with distinct changes in macrophage behavior following cardiac injury [2], where cells that once supported tissue regeneration begin to contribute to tissue fibrosis. We have previously shown significant alterations to the extracellular matrix (ECM) as a function of developmental age and that these changes significantly impact cardiac cells including cardiomyocytes [3] and cardiac fibroblasts [4]. The purpose of this study was to establish the unique macrophage response to ECM derived from neonatal or adult rat hearts and the ability of this response to affect other cells in the cardiac environment.

Materials and

Methods: : Cardiac tissue from neonatal and adult rats was decellularized and enzymatically digested by pepsin to solubilize. Extracellular matrix peptides were then allowed to adsorb onto tissue culture plastic overnight at a concentration of 100μg/cm2. Murine RAW 267.4 bone-marrow derived macrophages were seeded onto ECM-coated tissue culture plastic or a non-coated control and observed for 24 hours. Media samples were taken periodically to track cytokine secretion. After 24 hours, cells were analyzed for proliferation, morphology, and phenotype.

Results, Conclusions, and Discussions:: Macrophages grown on neonatal ECM display differences in cell proliferation, morphology, and surface marker expression compared to cells grown on adult ECM (Fig. 1a). Cell numbers were significantly higher (p = 0.0064) after 24 hours when grown on neonatal ECM as compared to adult ECM or the no-ECM control (Fig. 1b). Immunofluorescent staining of the mitosis marker Ki-67 (Fig. 1c) confirmed that cells grown on neonatal ECM showed higher levels of proliferation than other groups (p = 0.008). This increase in proliferation is consistent with results previously found in the lab for other cell types, including cardiomyocytes [3]. The increase in proliferation associated with neonatal ECM may play a role in the local proliferation of tissue resident macrophages following injury in neonatal animals not found in adult tissues. Additionally, cells grown on neonatal ECM also showed a larger range in size and shape compared to other groups (Fig. 1d and e). A decrease in circularity in cells grown on both adult and neonatal ECM compared to the control group (Fig. 1g) indicates an increase in cell activation, which is consistent with the literature. Cell phenotype will be further assessed in future experiments using immunofluorescent staining of inflammatory and anti-inflammatory markers, such as CD86 and CD206, respectively. Finally, cells grown on neonatal ECM show a more robust, but delayed, cytokine release profile (p = 0.0202), supporting an increase in activation of cells grown on neonatal ECM (Fig. 1f). More cytokine release may indicate an increased ability of macrophages to influence other cell types, which will be the focus of future research. The cellular mechanisms through which these differences in macrophage behavior are influenced by ECM will also be investigated. These findings support the use of neonatal cardiac ECM as a therapeutic material for modulating the immune response.

Acknowledgements and/or References (Optional):: [1] Porrello, E. R., Mahmoud, A. I., Simpson, E., Hill, J. A., Richardson, J. A., Olson, E., and Sadek, H. A. (2011) Transient regenerative potential of the neonatal mouse heart. Science, 331 (6020): 1078-1080. Doi.org/10.1126/science.1200708.
[2] Aurora, A. B., Porrello, E. R., Tan, W., Mahmoud, A. I., Hill, J. A., Bassel-Duby, R., Sadel, H. A., and Olson, E. N. (2014) Macrophages are required for neonatal heart regeneration. Journal of Clinical Investigations, 124 (3), 1382-1392. Doi.org/10.1172/JCI72181.
[3] Williams, C., Quinn, K. P., Georgakoudi, I., and Black, L. D. (2013) Young developmental age cardiac extracellular matrix promotes the expansion of neonatal cardiomyocytes in vitro. Acta Biomater, 10 (1): 194-204. Doi.org/j.actbio2013.08.037.
[4] Perreault, L. R., Le, T. T., Oudin, M. J., and Black, L. D. (2021) RNA sequencing indicates age-dependent shifts in the cardiac fibroblast transcriptome between fetal, neonatal, and adult developmental ages. Physiological Genomics, 53 (10), 414-429. Doi.org/10.1152/physiolgenomics.00074.2021.