Introduction/Aim: Pulmonary arterial hypertension (PAH) is a rare and fatal disease, and there is limited knowledge of the precise molecular signalling changes which lead to the endothelial cell abnormality that is a hallmark of this disease. Endothelial progenitor cells (EPCs) contribute to angiogenesis, and are also known to be altered in patients with PAH. How these cells exert their effect is unknown, however exosomes (Exo) are thought to contribute. We will develop and optimise the culture of EPCs from peripheral blood samples of PAH patients and the technique for Exo isolation and characterisation. We will compare the expression of known endothelial surface markers on these Exo’s.
Methods: EPCs were isolated and cultured from both PAH and control patient’s peripheral blood (15mL). Following EPC expansion and characterisation, cultures were serum starved for 48 h and the supernatant removed for centrifugal isolation of Exo’s. These were characterised using western blot and TEM, and assessment of surface marker expression on both Exo’s and EPCs was conducted via western blot.
Results: Culture of PAH peripheral blood derived EPCs was developed. A standard protocol to isolate, visualise via TEM and characterise Exo’s from both PAH and control EPCs was established. Furthermore, endothelial markers such as CD31, VEGFR2, CD146, Endoglin and VE-Cadherin were found to be expressed on the surface of these Exo’s. Preliminary results indicate there is significantly reduced expression of VEGFR2 on PAH derived Exo’s compared to control EPCs and Exo’s.
Conclusions: We have successfully cultured EPCs from 15mL of PAH patient peripheral blood as well as isolate and characterise Exo’s from these EPCs. Surface marker analysis of Exo’s revealed expression of endothelial surface markers with preliminary results indicating differential expression between control and PAH derived Exo’s. Further analysis of expression differences between control and PAH derived Exo’s is currently underway.