Extracellular vesicles (EVs) are membrane enclosures released by eukaryotic cells that carry bioactive molecules and serve to modulate biological responses in recipient cells. Both increased EV release and altered EV composition are associated with the development and progression of many pathologies including cancer. Hypoxia, a feature of rapidly growing solid tumours, increases the release of EVs. However, the molecular mechanisms remain unknown. The hypoxia inducible factors (HIFs) are transcription factors that act as major regulators of the cellular adaptations to hypoxia. Here, we investigated the requirement of HIF pathway activation for EV release in Human Embryonic Kidney Cells (HEK293). Time course experiments showed that EV release increased concomitantly with sustained HIF1α and HIF2α activation following the onset of hypoxia. shRNA mediated knock-down of HIF1α but not HIF2α abrogated the effect of hypoxia on EV release, suggesting HIF1α is involved in this process. However, stabilization of HIF proteins in normoxic conditions through: (i) heterologous expression of oxygen insensitive HIF1α or HIF2α mutants in normoxic cells or (ii) chemical inhibition of the prolyl hydroxylase 2 (PHD2) repressor protein, did not increase EV release, suggesting HIF activation alone is not sufficient for this process. Our findings suggest HIF1α plays an important role in the regulation of EV release during hypoxia in HEK293 cells, however other hypoxia triggered mechanisms likely contribute as stabilization of HIF1α alone in normoxia is not sufficient for EV release.

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

Mesenchymal stromal cell (MSC)-derived extracellular vesicles (EVs) have emerged as novel tools in regenerative medicine. Angiogenesis modulation is widely studied for the treatment of ischaemic diseases, wound healing, and tissue regeneration. Here, we have shown that EVs from human umbilical cord-derived MSCs can affect VEGFR2 signalling, a master regulator of angiogenesis homeostasis, via altering the phosphorylation of AKT. This translates into an inhibition of apoptosis, promoting exclusively cell survival, but not proliferation, in human microvascular endothelial cells. Interestingly, when comparing EVs from normoxic cells to those obtained from hypoxia (1% O2) preconditioned cells, hypoxia-derived EVs appear to have a slightly enhanced effect. Furthermore, when studied in a longer term endothelial-fibroblast co-culture angiogenesis model in vitro, both EV populations demonstrated a positive effect on vessel formation, evidenced by increased vessel networks with tubes of significantly larger diameters. Our data reveals that EVs selectively target components of the angiogenic pathway, promoting VEGFR2-mediated cell survival via enhancement of AKT activation. Our data show that EVs are able to enhance specific components of the VEGF signalling pathway and may have therapeutic potential to support endothelial cell survival.

Tracking the biodistribution of cell therapies is crucial for understanding their safety and efficacy. Optical imaging techniques are particularly useful for tracking cells due to their clinical translatability and potential for intra-operative use to validate cell delivery. However, there is a lack of appropriate optical probes for cell tracking. The only FDA-approved material for clinical use is indocyanine green (ICG). ICG can be used for both fluorescence and photoacoustic imaging, but is prone to photodegradation, and at higher concentrations, undergoes quenching and can adversely affect cell health. We have developed novel near-infrared imaging probes comprising conjugated polymer nanoparticles (CPNs™) that can be fine-tuned to absorb and emit light at specific wavelengths. To compare the performance of the CPNs™ with ICG forin vivocell tracking, labelled mesenchymal stromal cells (MSCs) were injected subcutaneously in mice and detected using fluorescence imaging (FI) and a form of photoacoustic imaging called multispectral optoacoustic tomography (MSOT). MSCs labelled with either ICG or CPN™ 770 could be detected with FI, but only CPN™ 770-labelled MSCs could be detected with MSOT. These results show that CPNs™ show great promise for tracking cellsin vivousing optical imaging techniques, and for some applications, out-perform ICG.