Using a Cre-loxP based method, I have been able to visualize transfer of EVs within human vascular cells, from PASMCs (donor) to PAECs (recipient). This has provided me with a potent in vitro tool to study contribution of EV-mediated cell:cell communication within the human vasculature. In fact, I have been able to apply this novel methodology to analyse the alteration of this EV-mediated transport and translation of transported mRNA by recipient cells in an in vitro model of pulmonary hypertension, testing the effect of different stimuli related with PAH. This has led to demonstrate an enhancement of this transport from PASMCs to PAECs with excessive TGF-β signalling, particularly in response to TGF-β1, as well as in response to IL1-β. These results highlight the biological relevance of PASMC-to-PAEC EV-mediated communication in the development of PAH. My results also prove that the enhancement observed is mainly due to an increased uptake of PASMC-EVs by recipient PAECs in response to the aforementioned stimuli.
We then aimed to analyse the cargoes enclosed in PASMC-EVs, which might therefore be transported into PAECs, taking special interest to lncRNAs. For that purpose we performed RNA-Seq analysis, which for such a scarce sample as EVs from primary cells it was very challenging. Unfortunately, the representation of lncRNAs in EVs was very low in percentage compared to the cellular RNA, which hampered the differential analysis resulting in very low number of lncRNAs. A great enrichment of FLJ16779 lncRNA (2929.3-fold) in PASMC-EVs was confirmed that pointed to a lncRNA very specifically expressed by EVs. Further analyses with another lncRNA found upregulated in PASMCs with TGF-β1-treatment, myoslid, showed enrichment in PASMC-EVs and therefore I conducted gain-of-function experiments to explore the specificity of its sorting into PASMC-EVs. After overexpression of myoslid in PASMCs, a very specific sorting into PASMC-EVs was observed, since fold-changes were 12.1 and 25562.1 respectively. Further experiments will be conducted to define the importance of EV-mediated transport of mysolid lncRNA from PASMCs to PAECs.
Since lncRNA data was not extensive, we sought to analyse protein-coding RNAs to expand the utility of the RNA-Seq performed. Surprisingly, PASMC-EVs in normal conditions were enriched in the transcription factor Zeb1, related to EndoMT, and two TGF-β ligands: GDF11 and TGF-β3. This will mean that the PASMC-EVs have the potential to induce effects on PAECs in normal conditions, since previous experiments had proven that transport of this EVs and translation of their mRNA occurs in normal conditions on recipient PAECs. Moreover, we observed enhanced transport and translation when PAECs were activated by PAH-related stimuli. Therefore, these cargoes could contribute to induce EndoMT and excessive TGF-β signalling on PAECs upon uptake of PASMC-EVs and play a crucial role in PAH. Analysis of the EndoMT marker SMA in green cells (do take up PASMC-EVs) vs. red cells (don’t take up PASMC-EVs) proved induction of EndoMT in recipient cells, thus demonstrating the proposed mechanism.
Differential analysis of PASMC-EVs with or without TGF-β1 stimulation showed 95 RNAs differentially expressed with treatment. GO analysis of these cargoes pointed to actin and ECM remodelling, thus suggesting involvement in vascular remodelling and EndoMT. We further validated 2 of these, palladin and bHLHE40, both related to EMT, a similar process to EndoMT. This reinforced a role for PASMC-EVs in PAH progression in a context of excessive TGF-β signalling, which underlies most cases of pulmonary hypertension.
