Fig. 5

EV-mediated transfer of mRNA from HPASMC to HPAECs results in protein translation in vitro. a, HPAECs in culture were treated 20 h with 5 × 10³ HPASMC-EVs/cell labelled with PKH67. Then, immunocytochemistry for CD31 was carried out. Many CD31⁺ HPAECs were shown to bind and take up HPASMC-EVs. b, Optimised Cre-loxP method for monitoring EV transfer for the first time applicable to in vitro primary cells with limited lifespan. A lentiviral vector was used to transduce donor cells (HPASMC) assuring high efficiency of transgene expression. Recipient cells (HPAECs) were transduced with the original lentiviral reporter construct at a low MOI = 0.1 to avoid background recombination and sorted in a FACS Aria Fusion sorter (BD Biosciences) to provide 100% Reporter+ cells. c, Amount of Cre mRNA quantified by qRT-PCR on EVs from HPASMC Cre⁺ and compared to control HPASMCs. Average fold-change above 2 × 10⁵ showed clear induction of the sorting of Cre mRNA into HPASMC-EVs. d, Co-cultures of HPASMC Cre⁺ and HPAECs Rep⁺ showed specific recombination on HPAECs switching into green fluorescence, as opposed to negative control HPASMC Null+: HPAECs Rep+ cells co-cultures. e, Quantification of Cre-induced recombination by FACS showed significant increase in co-cultures as opposed to control (HPAEC Rep⁺ only) and HPASMC Null⁺: HPAECs Rep⁺ cells co-cultures. f, The markers SMA and collagen III were quantified by flow cytometry at day 7 after HPASMC Cre⁺: HPAECs Rep⁺ co-culture. Differential analysis of HPAECs that take up HPASMC-EVs (eGFP⁺) vs. HPAECs that do not (DsRed⁺ eGFP⁻) showed a significant increase of SMA (13-fold, pvalue = 0.016) in eGFP⁺ cells and a mild close-to-significant increase of collagen III (1.06-fold, pvalue = 0.087). Data are presented as mean ± SEM. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 (Student’s t-test). All procedures were carried out with n = 3 biological replicates. Scale bars 4x = 300 μm; 10x = 120 μm; 20x = 60 μm. SMA: smooth-muscle actin.