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Digital archive of theses discussed at the University of Pisa


Thesis etd-12162019-104617

Thesis type
Tesi di dottorato di ricerca
Thesis title
Elucidating the role of Extracellular Vesicles on Pulmonary Arterial Smooth Muscle Cells - Pulmonary Arterial Endothelial Cells cross-talk in Human Pulmonary Arterial Hypertension using novel cutting-edge technologies to image functional cargo transfer
Academic discipline
Course of study
tutor Prof. Celi, Alessandro
  • Cre-loxP
  • EVs
  • Extracellular Vesicles
  • PAH
  • Pulmonary Hypertension
  • RNAseq
  • TGFB1
Graduation session start date
Pulmonary arterial hypertension (PAH) is a pathology characterised by acute remodelling of distal pulmonary arteries, right ventricular dysfunction, and increased pulmonary vascular resistance that promotes heart failure. Additionally, heritable PAH is caused by a mutation that results in impairment of the TGFβ superfamily signalling pathway. Previously considered as a largely untreatable disease, recent studies have utilised TGFβ1 treatment of Human Pulmonary Arterial Smooth Muscle Cells (HPASMCs) to mimic in vitro the mechanisms underlying PAH disease. Although extracellular vesicles (EVs) have been shown to influence the vascular environment, their role remains obscure for many pathologies. Recent work in Prof. Baker’s laboratory demonstrated that EVs are involved in communication within vascular cells during PAH.
The aim of this project has therefore been to try and assess whether EVs from HPASMCs treated with an excess of TGFβ1 are involved in transporting functional cargo to Human Arterial Endothelial Cells (HPAECs) and whether this cargo may affect the latter within the vascular microenvironment of PAH.
In order to demonstrate the transfer of mRNA and its translation into protein during EVs uptake, a Cre-LoxP method optimised for its use in primary cell cultures was assessed. With this method, we were able to determine whether HPAECs had taken up EVs re-leased by HPASMCs during PAH-simulated cell co-cultures. The method is as simple the-oretically as difficult to reproduce practically. The method´s design is to enclose Cre mRNA upon donor HPASMC EVs while recipient cells (HPAECs) show whether Cre+ EVs have been taken up by switching from red to green fluorescence. Fluorescence-activated cell sorting of eGFP out of DsRed positive cells (ratio of 1.53±0.26%) demonstrated that using our Cre-LoxP system we could observe the transfer and translation of Cre-mRNA into protein from HPASMC-EVs to HPAECs. Interestingly, this transfer was related to TGFβ1 treatment which resulted in the marked increase of EVs uptake from HPAEC but not an increase in the number of EVs released by HPASMCs.
Single-stranded low-input RNA-Seq was used to characterise HPASMC-EVs´ cargoes. The cargoes present in EVs from Control and TGFβ1 treated HPASMCs were also been analysed, measuring 2417 differentially expressed transcripts in HPASMC-EVs. Among these, a subset of 759 RNAs was found significantly enriched, in Control EVs when compared to their donor cells. Particularly interesting was the finding of overexpressed GDF11, TGFβ3 and Zeb1 transcripts, that was further investigated. Furthermore, EVs from TGFβ1 treated cells showed 90 differential transcripts when compared to Control EVs. Gene Ontology Enrichment Analysis showed that these transcripts were typically associated with cellular differentiation, migration, and response to wounding: all mechanisms associated with PAH development and/or PAH-related EndMT. Some of these genes involved in EndMT have been validated also by means of qRT-PCR, such as palladin and bHLHE40 that were found over-represented in HPASMC-EVs.
Preliminary experiments showed that, after treating HPAECs with HPASMC-EVs, their cellular phenotype changed clearly at visual examination under the microscope; although, this behaviour needs to be investigated further. In summary, the present results illustrate the potential role for EVs during PAH pathogenesis. The in vitro Cre-LoxP system optimised for primary cells showed that HPASMC-EVs can release functional cargo. The transcriptomic data showed differential regulation of several transcripts that are critical for the remodelling of the vasculature during PAH development, as pilot experiments on HPAECs treated with HPASMC-EVs pointed out. However, more research on these preliminary results needs to be undertaken to better define the association between HPASMC-EVs uptake and PAH.