Using Patient-Derived Stem Cells to Study HIF2 Regulation in Pulmonary Hypertension

Erythrocytosis is a condition in which an abnormally high number of red blood cells (RBCs) are present in the blood. In a healthy person, the production of RBCs are tightly regulated by erythropoietin (EPO), which is controlled by a member of the hypoxia inducible factor (HIF) family, HIF2A. Under normoxic conditions, HIF2A is hydroxylated by prolyl hydroxylase domain protein (PHD), targeting it for degradation. Under hypoxic conditions, this modification of HIF2A is inhibited to stabilize and promotes downstream expression of a variety of target genes, including EPO. As a result of a gain-of-function mutation in the oxygen-sensitive transcription factor HIF2A, a specific group of patients demonstrated erythrocytosis and developed pulmonary hypertension (PH). One main characteristic of PH is endothelial dysfunction, leading to structural changes of pulmonary vasculature. Mouse model with gain-of-function HIF2A mutation demonstrated erythrocytosis and developed PH similar to human patients. However, to date, it is unknown how the HIF2A+ mutation leads to PH, clear of the effects of erythrocytosis. Using human induced pluripotent stem cell (hiPSC) line derived from an erythrocytosis patient and an optimized endothelial cell differentiation protocol we created a human in vitro system to study HIF2A+ vascular cells. We found that HIF2A+ hiPSC-derived ECs (VEcad-positive cells) can mature to functional endothelial cells–expressing mature markers: von Willebrand factor and endothelial nitric oxide synthase are capable of lectin binding and acetylated low-density lipoprotein uptake. Real-time PCR results show that HIF2A mRNA expression is specifically up-regulated in hiPSC-derived ECs. Endothelin-1 (EDN1) expression is significantly higher in HIF2A+ hiPSC-derived ECs compared to the ECs derived from health donor hiPSCs, correlating with the HIF2A gain-of-function mutation. In addition, the expression of ECM proteins including laminin and fibronectin is higher in HIF2A+-ECs compared to the control. We speculated that these differences in gene expression are caused by overactive HIF2A expression, which causes endothelial dysfunction in PH.