Figure 3.

SFLLRN induces expression of IL‐8 mRNA and protein in late EPC. (A) Early and late EPC were cultured in endothelial basal medium‐2 (EBM2) for 24 hrs and IL‐8 was quantified in the medium by ELISA. IL‐8 (ng/10⁶ cells) was at 312 ± 28 in early EPC and 338 ± 3 after SFLLRN treatment (P= 0.46). IL‐8 in late EPC from adult blood averaged 15 ± 2 ng/10⁶ cells at baseline versus 273 ± 80 ng/10⁶ cells after SFLLRN treatment (*P= 0.014). In late EPC from cord blood, IL‐8 was at 110 ± 7 ng/10⁶ cells versus 471 ± 196 ng/10⁶ cells after SFLLRN treatment (*P= 0.034). (B) Effect of PAR‐1 activation on mRNA expression of IL‐8 and its receptors CXCR1 and CXCR2 by early EPC from adult blood (AB) and late EPC from AB and cord blood (CB). Gene expression was considered relevant when the Ct value (representing the threshold cycle number at which PCR products become detectable) was below 35. EPC were stimulated with SFLLRN 75 μM for 4 hrs after 16 hrs of serum and growth‐factor privation (EBM2). mRNAs were measured by RTQ‐PCR and normalized to the ubiquitous gene TBP mRNA. Normalized mRNA levels were compared between stimulated and untreated control cells (arbitrarily = 1). Data are means ± S.E.M. of at least three independent experiments. (C) Time‐dependent fold‐increase in IL‐8 gene expression upon PAR‐1 activation by SFLLRN in CB late EPC. Serum‐starved CB late EPC were kept unstimulated or stimulated for 1, 4, 8, 12, 24 or 48 hrs with SFLLRN 75 μM. MRNAs were measured by RTQ‐PCR and normalized to the ubiquitous gene TBP mRNA. Normalized mRNA levels were compared between stimulated and untreated control cells (arbitrarily = 1). Data are means ± S.E.M. of at least three independent experiments.