Dewey, Marley (University of Pittsburgh )


Introduction. Matrix-bound nanovesicles (MBV) are nanometer-scale extracellular vesicles secreted by cells and found embedded within the extracellular matrix (ECM)[1]. MBV are similar to exosomes in size and shape, but MBV have distinctly different lipid profiles and RNA cargo[2]. MBV have demonstrated the ability to induce an M2-like pro-healing macrophage phenotype, promote neuronal stem cell differentiation, and suppress pro-inflammatory astrocyte signaling in optic nerve repair[3,4]. MBV have the potential to be used as a therapeutic and diagnostic tool, much like exosomes; however, an understanding of MBV biogenesis is lacking and hence their full potential depends on developing an understanding of the mechanisms of action. Our goal is to determine MBV biogenesis by answering the questions of the intracellular biogenesis of MBV, the mechanism of transport and binding to the ECM, and the mechanisms that govern their production.

Methods. To evaluate the intracellular origin of MBV, we used fluorescent lipid dyes to stain for lipids specific to cellular compartments. MBV and exosomes were isolated from the same cell source (fibroblasts) and were stained with lipids related to various cellular compartments (ie. Golgi) to determine if MBV or exosomes stained positively for these lipids and if we could live-cell image MBV production. To evaluate how MBV bind to the ECM we assess ECM-related surface markers on both MBV and exosomes and use high-resolution imaging to visualize vesicle location related to collagen production. Finally, we assessed the mechanisms that govern MBV production by sequentially deleting ESCRT-independent and -dependent subunits, a pathway in which exosomes use for their production, and assessing the impact of this on MBV production and collagen formation.

Results. We report a process for isolating MBV and exosomes from the same cell source, exosomes from the liquid phase and MBV from the deposited ECM. Ongoing work will be presented on the lipid staining differences between MBV and exosomes, and we expect that MBV and exosomes will have staining differences, as previous work demonstrates MBV have different lipid profiles from exosomes[2]. Ongoing data will be presented on ECM-binding surface proteins on MBV and exosomes, and whether MBV bind to collagen fibrils during or after collagen production. Finally, we show that GW4869, an ESCRT-independent pathway inhibitor, does not inhibit collagen and MBV production, possibly indicating that MBV do not use the ESCRT-independent process for production. We will further present the impact of ESCRT-dependent deletions on MBV and collagen production.

Conclusions. The present work will expand on our knowledge of a new class of extracellular vesicle, MBV. By understanding the mechanisms of their production, the origin of their production, and how they bind to the ECM, this will allow us to better realize their theranostic potential.

[1] Huleihel, L. et al., Sci Adv. 2, e1600502 (2016).
[2] Hussey, G. S. et al., Sci Adv. 6, eaay4361 (2020).
[3] Huleihel, L. et al., Tissue Eng Part A. 23, 1283-1294 (2017).
[4] van der Merwe, Y. et al., Sci Rep. 9, 3482 (2019).
[5] Laulagnier, H. et al., Blood Cells Mol Dis, 35, 116-21 (2005).


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