Iron Containing Compound Stimulates Expression of Pulmonary Hypertension Promoting Factor PlGF

Abstract 900

We have previously reported elevated plasma levels of placental growth factor (PlGF) and endothelin-1 (ET-1) in patients with sickle cell disease with high estimated pulmonary artery systolic pressure, a marker of pulmonary hypertension, and gene transfer experiments in mice document that PlGF stimulates ET-1 expression and pulmonary hypertension. Among other markers, PlGF in SCD subjects is correlated with markers of excessive iron burden, including serum ferritin and transferrin, markers that have been previously associated with pulmonary hypertension in SCD in many studies. We therefore hypothesized that iron stimulates expression of PlGF. Because tissue postnatal expression of PlGF in vivo is restricted primarily to early erythroid cells, we chose K562 erythroleukemia cells as a cell culture model. We find that heme-bound iron (hemin) induces PlGF mRNA robustly in a dose-dependent and time-dependent fashion. The PlGF transcript rises robustly within hours of hemin stimulation, and reaches levels of 40 to 80-fold over baseline in a real time PCR assay. Heme analog compound, mesoporphyrin without iron fails to induce PlGF, but as a control induces hemeoxygenase-1 mRNA. Iron plus mesoporphyrin induces PlGF strongly, indicating a critical requirement for iron in inducing PlGF transcript. In promoter-reporter luciferase constructs transfected into K562 cells, hemin induces the human PlGF promoter significantly, and a minimal promoter construct contains binding sites for erythroid Kruppel-like factor (KLF-1). KLF-1 mRNA levels rise in hemin-stimulated K562 cells in parallel with PlGF, and in peripheral blood mRNA from SCD subjects and healthy controls, the level of KLF-1 transcript correlates closely with PlGF transcript (r=0.82, p<0.0001). Additional studies of KLF-1 are under way.

Our results indicate for the first time a specific mechanistic pathway induced by iron that is linked in humans with SCD and in mice to vasculopathy and pulmonary hypertension. Rather a simple epiphenomenon of frequent transfusion or source of nonspecific oxidative stress, iron may trigger a KLF-1/PlGF/ET-1 linear mechanistic pathway to stimulate pulmonary hypertension. This suggests that PlGF levels are linked to the iron overload in patients with SCD. Further details in this pathway are under investigation.