SnRNA sequencing defines signaling by RBC-derived extracellular vesicles in the murine heart.

Nedyalka Valkov,Robert Kitchen,Priyanka Gokulnath,Srimeenakshi Srinivasan,Saumya Das,Vasilis Toxavidis,Chunyang Xiao,Ashish Yeri,Guoping Li,Nathan R Tucker,John Tigges,Avash Das,Ionita Ghiran,Olivia Ziegler,Aushee Khamesra,Shulin Lu,Rodosthenis Rodosthenous,Stefan Momma,Patrick T Ellinor ,Mark Chaffin ,Louise C Laurent ,Getúlio Pereira De Oliveira Júnior ,Ane M Salvador ,Ivan-Maximilano Kur

doi:10.26508/lsa.202101048

Abstract

Extracellular vesicles (EVs) mediate intercellular signaling by transferring their cargo to recipient cells, but the functional consequences of signaling are not fully appreciated. RBC-derived EVs are abundant in circulation and have been implicated in regulating immune responses. Here, we use a transgenic mouse model for fluorescence-based mapping of RBC-EV recipient cells to assess the role of this intercellular signaling mechanism in heart disease. Using fluorescent-based mapping, we detected an increase in RBC-EV-targeted cardiomyocytes in a murine model of ischemic heart failure. Single cell nuclear RNA sequencing of the heart revealed a complex landscape of cardiac cells targeted by RBC-EVs, with enrichment of genes implicated in cell proliferation and stress signaling pathways compared with non-targeted cells. Correspondingly, cardiomyocytes targeted by RBC-EVs more frequently express cellular markers of DNA synthesis, suggesting the functional significance of EV-mediated signaling. In conclusion, our mouse model for mapping of EV-recipient cells reveals a complex cellular network of RBC-EV-mediated intercellular communication in ischemic heart failure and suggests a functional role for this mode of intercellular signaling.

Highlights

Extracellular vesicles (EVs) are cell-derived membranous structures (100–1,000 nm in diameter) comprising exosomes and microvesicles [1]
Fluorescence microscopy of the peripheral blood smear and cell-free plasma (CFP) from EpoRCre/mTmG double transgenic mice confirmed that all RBCs in the circulation and a substantial fraction of the EVs in plasma were membrane-targeted EGFP (mGFP)+, as expected (Fig 1B)
Nano-flow cytometry of the plasma from EpoR-Cre/mTmG mice confirmed the presence of mGFP+ EVs, whereas plasma from mTmG mice showed only mTd+ EVs suggesting a large contribution of GFP+-EVs from RBCs (Fig 1C) some contribution from platelets cannot be excluded

Summary

Introduction

Extracellular vesicles (EVs) are cell-derived membranous structures (100–1,000 nm in diameter) comprising exosomes and microvesicles [1]. Most of our understanding about EV function comes from studies using EVs derived either from cell culture conditioned media or biological fluids, and their subsequent administration in animal models to assess functional changes. This approach is less likely to reflect their in vivo composition and endogenous functions. We have previously shown that functional Cre mRNA can be packaged in exosomes released by Cre recombinase–expressing cells and transferred to EV-recipient reporter cells, subsequently mediating Cre-dependent recombination to allow expression of a reporter [4] This system has allowed us to map targets of hematopoietic cell (HC) derived EVs at baseline and in a model of peripheral inflammation. Our work has demonstrated that neuronal activity can influence the uptake of these EVs [9], suggesting an important role for the state of the recipient cell in this mode of signaling

Methods

Results

Conclusion