Extracellular vesicles released by fibroblasts undergoing H-Ras induced senescence show changes in lipid profile.

Sandra Buratta,Nico Mitro,Silvia Caponi,Stefano Giovagnoli,Lorena Urbanelli,Carla Emiliani,Donatella Caruso,Krizia Sagini,Daniele Fioretto,Giovanni Camussi

doi:10.1371/journal.pone.0188840

Abstract

Cells release extracellular vesicles (EVs) in their environment and cellular lipids play an important role in their formation, secretion and uptake. Besides, there is also evidence that EV transferred lipids impact on recipient’s cell signaling. Cellular senescence is characterized by a state of permanent proliferation arrest and represents a barrier towards the development of neoplastic lesions. A peculiar feature of senescence is the release of many soluble factors, the so-called Senescence-Associated Secretory Phenotype, which play a key role in triggering paracrine senescence signals. Recently, evidences have suggested that this phenotype includes not only soluble factors, but also EVs. To identify lipid signatures associated with H-Ras-induced senescence in EVs, we expressed active H-Ras (H-RasV12) in human fibroblasts and investigated how it affects EV release and lipid composition. An enrichment of hydroxylated sphingomyelin, lyso- and ether-linked phospholipids and specific H-Ras-induced senescence signatures, e.g. sphingomyelin, lysophosphatidic acid and sulfatides, were found in EVs compared to cells. Furthermore, H-RasV12 expression in fibroblasts was associated with higher levels of tetraspanins involved in vesicle formation.

Highlights

Cells can exchange information through the release of small vesicular bodies called extracellular vesicles (EVs) [1,2,3]
H-RasV12 was expressed in human dermal fibroblasts (HuDe) by transfection followed by pharmacological selection, using empty vector as control
As EVs are usually enriched in specific proteins, we performed immunoblotting using as positive markers CD9, CD63 and CD81 tetraspanins, Alix and Tsg101 (ESCRT machinery proteins involved in vesicle biogenesis), Rab5b, LAMP2 and flotillin 1, in agreement with guidelines [32]

Summary

Introduction

Cells can exchange information through the release of small vesicular bodies called extracellular vesicles (EVs) [1,2,3]. All cell types investigated so far have been found to release EVs. It is accepted that cells can release several types of EVs, characterized by heterogenous size, biogenesis and cargo [4]. EVs can originate either from the plasma membrane (microvesicles or ectosomes) or from the endosomal system (exosomes). Microvesicles show a wide size distribution, ranging from a few dozens of nanometers to a few micrometers, whereas vesicles.

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Conclusion