Periodic Reporting for period 1 - EV-LNC (Extracellular vesicle-mediated delivery of long non-coding RNA: Implications for vascular repair and regeneration)
Reporting period: 2017-08-01 to 2019-07-31
Methodology: For imaging EV transfer among vascular cells, we will use a pioneer approach based on Cre-loxP recombination which results in a fluorescent colour switch of cells upon EV uptake. Cells will then be sorted according to fluorescence by flow cytometry and analysed by cutting-edge proteomics and bioinformatics, in order to elucidate intercellular signalling triggered by EV transfer. The lncRNAs present in EVs will be analysed by RNA-Seq. Mechanistic insight of enriched lncRNAs in EVs will be evaluated using gain- and loss-of function approaches in vascular cells using lentiviral vectors and GapmeRs/siRNAs, respectively. Integration of all these analyses will provide key information to define implications of EV-mediated delivery of lncRNA for vascular repair and regeneration.
• PASMCs communicate with PAECs via EVs and the latter take up and translate RNA cargo to synthesize proteins.
• PASMC-to-PAECs EV-mediated communication is enhanced by TGF-β1 and IL1-β possibly due to increased uptake of PASMC-EVs activated PAECS. Since these cytokines are increased in pulmonary hypertension, this communication may be enhanced during PAH.
• PASMC-EVs are enriched in the transcription factor Zeb1, related to EndoMT, and two TGF-β ligands: GDF11 and TGF-β3, at the RNA level. Zeb1 at the protein level too.
• TGF-β1 treated PASMC-EVs increase their levels of paladin and bHLHE40 protein-coding RNAs, involved in EndoMT.
• PAECs that take up PASMC-EVs increase their levels of smooth-muscle actin, the most widely used EndoMT maker. Thus, PASMC-EVs can induce EndoMT on PAECs.
• PASMC-EVs are enriched in the lncRNA FLJ16779, which is very specific of PASMC-EVs.
• TGF-β1 treated PASMC-EVs overexpress mysolid lncRNA. Overexpressed mysolid is specifically sorted into PASMC-EVs and might be transported into PAECs.
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.
The EV-LNC project has generated a substantial amount of valuable data on the biological relevance of EV-mediated transfer of RNA from smooth muscle to endothelium in the pulmonary artery. Moreover, it has given evidence that this cell:cell communication may be enhanced during PAH and has put it in the spotlight as a novel contributor to physiology and pathology of the pulmonary artery. Although further studies are needed to unravel the molecular mechanisms underlying this, the data generated has an undoubted value for publication and as such, is under review in Cell Communication and Signalling. The results obtained set the basis for future studies addressing the impact of this cell:cell communication in the pathology of pulmonary hypertension and pave the way for modulating this transport with therapeutical purposes. The work performed and the networks established during the project will be exploited in the future to address these objectives.