Molecular Mechanisms of Hypoxia‐Induced Extracellular Vesicle Release

Abstract

Background Extracellular vesicles (EVs) are released by many cells and provide a mechanism for intercellular communication in autocrine and paracrine fashion. EVs of various sizes, namely microvesicles (ranging from 100–1000 nm) or exosomes (with sizes between 30–150 nm) are released by most eukaryotic cells. EVs are known to contain bioactive molecules including proteins lipids and nucleic acids including miRNAs and LNC RNAs that regulate gene expression and cell functions in recipient cells. For example, tumour released exosomes remodel the microenvironment allowing cancer cells to survive and thrive. Low oxygen (hypoxia) is known to increase the release of exosomes in cancer cells. Hypoxia leads to the activation of hypoxia inducible factor (HIF) transcription factors, considered master regulators of the hypoxic response. The objective of this study was to investigate whether hypoxia induced EV (exosome) release is dependent on hypoxia inducible factor (HIF) proteins. Methods We isolated exosomes and microvesicles by differential centrifugation of conditioned media of cells exposed to normoxia or hypoxia (1% Oxygen) for different lengths of time ranging from 1–24hrs. We determined, size distribution and concentration of vesicles by nanoparticle tracking analysis (NTA). Expression of HIF1 and HIF2 was monitored by western blot of cellular lysates. We expressed oxygen‐stable mutated HIF1 and HIF2 isoforms either alone or in combination and determined EV release by NTA. We used chemical inhibitors of dioxygenase (PHD) and von Hippel–Lindau (VHL) proteins, the two proteins involved in HIF degradation under oxygen conditions, to stabilize HIF expression in normoxic cells and analysed EV release and HIF expression. We also analysed EV release in a cell line lacking VHL and after a VHL knock‐in. Western blotting of cellular lysates was used to monitor HIFs, VHL, and VEGF proteins. NTA was used to determine EV concentration and size. ANOVA statistical analysis was performed to analyse the data. Results Hypoxia increased the amount extracellular vesicles in the conditioned media of all cells tested. The release of small vesicles (exosomes) was most increased following the exposure to hypoxia. HIF protein expression increased steadily during the time course of hypoxia, reaching a plateau after 2hrs following exposure. Expression of mutated (oxygen stable) HIF1 and HIF2 proteins in normoxic cells, either alone or in combination, did not increase exosome release. Chemical inhibition of PHD or VHL did not affect EV release. Exosome release was not different in VHL KD or KI cells. Conclusion these data suggest that hypoxia driven exosome release is HIF independent. Support or Funding Information Sponsored by Fundacion Ramon Areces

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