Biogenesis and release of endothelial extracellular vesicles: Morphological aspects

ElsevierVolume 245, January 2023, 152006Annals of Anatomy - Anatomischer AnzeigerAbstractBackground

Cell-cell communication through extracellular vesicles (EVs) including exosomes, microvesicles and apoptotic bodies has been shown to be important in physiological homoeostasis as well as pathological processes such as atherosclerosis. However, while the cellular machinery controlling EV formation and composition has been studied during the past decade, less is known about the morphological process of their formation and release.

Methods

Using different electron microscopic approaches including transmission-, scanning-, immun-, and serial block face electron microscopy we studied the morphogenetic events of EV formation and release. We analysed the different steps of EV formation and release in cultured myocardial endothelial (MyEnd) and aortic endothelial (AoEnd) cell lines under serum starvation and under inflammatory conditions.

Results

We show that in a narrow time frame, the number of active cells and microvesicle (MV) producing cells increased in dependence of time spent in cultivation and additional stimulation by TNF-α. However, MV secretion was a highly heterogeneous process which couldn´t be seen in all cells cultivated under the same conditions. Release of MVs could be observed all over the cells’ surface with no preferred release site. While no single specific microscopic approach applied was sufficient to provide a comprehensive analysis of EV biogenesis, we show that the limitations of one technique could be compensated by the qualities of the respective other applied techniques, thus enabling us to provide a detailed ultrastructural analysis of MV and exosome biogenesis. Surprisingly, exosome release in endothelial cells occurred via a yet undescribed process indicating that MVBs were incorporated into a novel distinct cellular compartment covered by fenestrated endothelium before exosome release. Lastly, we could show that TNF-α stimulation of AoEnd cells leads not only to the upregulation of CD44 in parental cells, but also to incorporation of CD44 into the membranes of generated MVs and exosomes.

Conclusions

Taken together, our data contribute to a better understanding of biogenesis and release of EVs. We conclude that under inflammatory conditions, EVs can mediate the transfer of CD44 from endothelial cells to target cells at distant sites including vessel wall cells and this could be a mechanism by which MVs may change the and thus contribute to the development and progression of atherosclerotic lesions.

Keywords

Extracellular vesicles

Microvesicles

Exosomes

Endothelial cells

Cell-cell communication

Atherosclerosis

AbbreviationsEVs

extracellular vesicles

MVBs

multi-vesicular bodies

ILVs

intraluminal vesicles

CVDs

cardiovascular diseases

MyEnd cells

myocardial endothelial cells

AoEnd cells

aortic endothelial cells

TNF-α

tumor necrosis factor-α

TEM

transmission electron microscopy

SEM

scanning electron microscopy

SBF-SEM

serial block-face scanning electron microscopy

CLSM

confocal laser scanning microscopy

View Abstract

© 2022 Published by Elsevier GmbH.

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