Fig 1.
Vector map, generation and purification of CD39 mRNA.
(A) Vector map of the pcDNA 3.3 plasmid containing the ENTDP-1/CD39 construct for mammalian expression. (B) Electrophoresis with a 1% agarose-TBE gel of the purified CD39 mRNA (1533 bp). eGFP mRNA (993 bp) was used as a loading control. 0.5–10 kB RNA ladder.
Fig 2.
Expression of CD39 induced by in vitro transfection of HEK293 cells and ECs with the generated CD39 mRNA.
Flow cytometric analyses of transfected HEK293 cells (A) human ECs (B) and immortalized porcine endothelial cells (PAE; C) with different CD39 mRNA concentrations. For control untreated and Lipofectamin-treated cells were used. The cells were labeled with a CD39-FITC antibody 24 h after transfection. Each experiment was performed at least 3 times. Data are given as means and SEM and were compared using one-way ANOVA (A & B) or RM-ANOVA (C) with Bonferroni´s multiple comparison test. ***p < 0.001. Representative fluorescence microscopic images of CD39 mRNA transfected (C, 2 μg CD39 mRNA) and untransfected (D) HEK293 cells.
Fig 3.
Overexpression of CD39 has no influence on the viability of the mRNA transfected HEK293 cells and the release of pro-inflammatory cytokines.
An MTT assay was performed after 24 h (A) and 48 h (B) to analyze the cell viability in HEK293 cells after transfection with CD39 mRNA. (C) Detection of various cytokines released from untransfected and transfected HEK293 cells with 0.5 μg and 1 μg CD39 mRNA using a Proteome Profiler TM Array. The supernatant of untreated and Lipofectamin-treated HEK293 cells were used as control. Data are given as means and SEM and were compared using one-way ANOVA with Bonferroni´s multiple comparison test; ns: not significant.
Fig 4.
The expressed CD39 protein in HEK293 cells and ECs is highly functional in hydrolyzing ADP.
CD39-transfected HEK293 cells (A) and human ECs (B) as well as untreated and Lipofectamin-treated cells were incubated with 200 μl ADP for 10 min at 37°C. Free phosphate was measured after incubating with 20 μl working solution in a plate reader at 620 nm absorbance. Data are given as means and SEM and were compared using RM-ANOVA with Bonferroni´s multiple comparison test. ***p < 0.001.
Fig 5.
Overexpression of CD39 in HEK293 cells and endothelial cells significantly decreases platelet activation in vitro.
CD39-transfected HEK293 cells (A) and EC cells (B) as well as untreated and Lipofectamin-treated cells were incubated with 20 μl ADP for 10 min at 37°C. Afterwards, the supernatants were incubated with PRP and platelet activation induced by non-hydrolyzed ADP was detected using an anti-CD62P-PE antibody in flow cytometry. Data are given as means and SEM and were compared using one-way ANOVA with Bonferroni´s multiple comparison test. **p < 0.01; ***p < 0.001.
Fig 6.
CD39 mRNA coating on PLGA slides significantly induces an expression in HEK293 cells.
Thermanox slides were coated with PLGA and CD39 mRNA/ Lipofectamin complexes. A coating of eGFP/ Lipofectamin was used as control. After incubation of the slides with HEK293 cells the expression of CD39 and eGFP was measured by flow cytometry after 24 h (A), 48 h (B) and 72 h (C). Data are given as means and SEM compared using one-way ANOVA with Bonferroni´s multiple comparison test. ***p < 0.001.
Fig 7.
The hypothetical effect of a CD39 mRNA-stent coating on endothelial cells.
Implantation of a CD39 mRNA-coated stent into an atherosclerotic blood vessel will result in transfection of endothelial cells, which in turn express the CD39 protein. The overexpression of the CD39 protein will result in augmented hydrolysis of ATP and ADP and hence may inhibit platelet activation and neointima formation as well as support anti-inflammatory processes.