Impact of cardiac health status on bone marrow cell secretome and myocardial repair
Authors: Ahmed S , Borrego I , Frobert A , Cook S , Giraud M N 
Published: May 2024

Abstract

Funding Acknowledgements
Type of funding sources: Public Institution(s). Main funding source(s): University of Fribourg Graduate assistant

Background
Restoring the damaged myocardium remains a significant clinical challenge. Administration of bioactive factors, exosomes, or cell secretomes has shown promising results in promoting angiogenesis, tissue repair, immunomodulation, and extracellular matrix remodelling, particularly for cardiovascular disease treatments. Bone marrow (BM) cells are an essential source of secretome-based therapies for myocardial infarction (MI). Harnessing the potential of the cellular secretome for therapeutic applications requires a comprehensive understanding of its composition, functions, and mechanisms of action. MI affects the entire hematopoietic hierarchy, from stem cells to myeloid progenitors in BM. The study investigates how the cardiac status healthy versus infarcted affects the BM cell compositions and the properties of their secretomes.

Methods
Bone marrow cells (BMC) were isolated from healthy male Lewis rats (hBMC) and infarcted one (iBMC) obtained 2 weeks post MI induced by coronary artery ligation and cultured for 2 weeks. Cells composition, particularly macrophages, was characterised using multiplex flow cytometry Cytek Aurora. (CD90, CD29, CD45. CD68, CD206, CD163, Arg1, CD80, CCR7, iNOS). Cell proliferation (Real-time cell analyser and EdU incorporation), metabolic assays (seahorse assays), PCR, MS proteomic, and potency assays for immunomodulatory properties were performed.

Results
Multiplex flow cytometry showed distinctions in the percentage of cells: CD34+ stem cells and hematopoietic CD45+ cells were significantly upregulated in hBMCs, while CD68+ cells were higher in iBMCs. Cell proliferation assay demonstrated that iBMCs had significantly lower proliferation rates compared to hBMCs. Cell Metabolism was also impacted, iBMCs showed increased basal respiration, ATP production, and maximal respiration compared to hBMCs. An increase in respiratory spare capacity in iBMCs suggests an increased potential for ATP production under increased energy demands. Relative to hBMC, iBMC significantly regulated the expression of genes involved in immune response, inflammation, angiogenesis, and ECM remodelling gm-csf, il-17, vcam1, stat6, mmp9, cam, arg1, mhcII, arg2 were downregulated and tlr2, cxcl10, il-6, timp1, rantes, mcp-1, vegf, il-1ra, il-1beta, mip-1alpha, tlr4, b2m, and mmp3 upregulated. Secreted proteins also varied. Finally, the secretome of iBMC showed an enhanced capacity to induce a phenotype switch of macrophages toward an anti-inflammatory profile.

Conclusion
Our study shows that MI significantly alters the properties of bone marrow cells, impacting their composition, proliferation, metabolism, gene expression and the immunomodulatory potential of the secretome. These findings highlight the importance of considering donor health status in developing restoring secretome-based therapies for cardiac repair and emphasise the need to incorporate potency assay of the biologics for a standardised therapy and quality control.