JSCDH-D-17-00019: Heme Promotes Pulmonary Hypertension in SCD by Inducing Secretion of Placenta Growth Factor from Erythroblasts via Oxidant Response

Background: Patients with sickle cell disease (SCD) have elevated plasma levels of placenta growth factor (PlGF) which promotes expression of the pulmonary vasoconstrictor endothelin-1 (ET-1) contributing to pulmonary hypertension, an important age-related and life-limiting complication of SCD. In SCD patients, markers of high iron burden are associated with the highest PlGF levels, leading us to hypothesize a mechanistic link between excessive iron and the induction of the PlGF protein. Methods: We have established in vitro models of heme-bound iron (hemin) stimulation of the PlGF promoter in human K562 cells and in primary human erythroid cells. With the use of real time PCR, we have assessed the transcriptional regulation of several genes involved in the NRF2 antioxidant response pathway as well as their contribution to PlGF and HO-1 transcriptional regulation. We have established a murine primary bone marrow cell culture model and an in vivo murine model of PlGF response to heme exposure. Results: Gene expression knockdown and small molecule inhibitor and activator experiments demonstrate a central role of NRF2 in activating the PlGF promoter in response to heme, supported by chromatin immunoprecipitation experiments. The PlGF promoter is robustly activated by a well characterized non-oxidative NRF2 agonist (Sulforaphane) and by the nonspecific oxidant hydrogen peroxide, while it is inhibited by an NRF2 inhibitor (Brusatol), by siRNA directed against NRF2 and by the nonspecific antioxidant N-acetyl cysteine. We also find that NRF2 activation regulates the transcript levels of several other NRF2 family members. PlGF induction overlaps with HO-1 regulation except that PlGF is apparently not regulated by BACH-1. Finally, we have confirmed that intravenous injection of heme in wild type mice induces PlGF mRNA expression in bone marrow, and secretion of PlGF protein into blood within three hours. This response is dramatically blunted in mice deficient in NRF2, further supporting the importance of NRF2 in heme induction of PlGF expression in vivo. Conclusions: Our results to date support a mechanism in which accelerated heme turnover in SCD promotes robust