Fig. 4.

Pharmacological interferences of CYP4A/20-hydroxyeicosatetraenoic acid (20-HETE) alter endothelial progenitor cell (EPC) stemness. Human cord blood-derived EPCs were harvested and cultured for 9 days before treating with dibromo-dodecenyl-methylsulfimide (DDMS; 10 μM) and 6,15-20-HEDGE (10 μM) (A), or 20-HETE (1 and 10 nM) (B) for an additional 7 days. DMSO was used as solvent control. Cells were then fixed and stained with anti-CD133 (1:500) and anti-CD34 (1:500) antibodies (indicative of EPC population with high stemness). Flow cytometry analysis was performed in day 16 culture to quantify the number of progenitor cells based on CD133⁺CD34⁺ cell surface marker (mean ± SD; n = 3; *P < 0.05 vs. solvent controls). In a parallel experiment, another batch of 9-day-old EPCs were treated in the presence and absence of 20-HETE (1 nM) for 4 h (C). Real-time PCR was carried out for gene expression of Oct 4, Sox2, and Nanog (mean ± SD; n = 3 in triplicates; *P < 0.05 vs. corresponding controls).