Relationship of iron to PlGF expression and death. (A) Elevated serum ferritin level is associated with higher mortality in SCD by ferritin tertile (n = 279; Kaplan-Meier plot, P < .0001; log-rank [Mantel-Cox] test, P < .0001; log-rank test for trend). (B) PIGF plasma concentration varies as a function of ferritin level, a recognized iron overload marker in SCD patients. (C) K562 cells were incubated with 20 µM hemin for 24 hours, and cells were harvested for immunoblot with anti-PlGF. Equivalent amount of protein from control and tested cell lysate were loaded into 4% to 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred onto nitrocellulose membrane, and blotted with antihuman PlGF. PlGF enzyme-linked immunosorbent assay was performed with medium and calculated per million cells. (D) Regular RT-PCR showed that PlGF expression is specifically upregulated by hemin. The oligonucleotides used for PCR span all 7 exons of PlGF mRNA (“Materials and methods”). (E-F) K562 cells were incubated with either indicated concentrations of hemin or same amount of vehicle, and cells were harvested and RNA extracted. cDNA was prepared with use of an Applied Biosciences kit, and quantitative PCR was performed. Time course and concentration dependence of (E,G) heme oxygenase 1 (HO-1) expression as a positive control and (F,H) PlGF expression in response to hemin.