Our studies pursue exciting new data supporting the hypothesis that the circulating monocyte (MON) and its derivatives, the MON-derived dendritic cell (MO-DC) and the MON-derived macrophage (MO-MØ) orchestrate a chronic innate immune response that underlies the progressive occlusive vascular pathology in pulmonary arterial hypertension (PAH). In Aim 1, we relate a reduction in BMPR2, the most frequently mutated gene in PAH, to an increase in the retroviral element HERV-K in MON. We extend new findings in MON suggesting that reduced BMPR2, through a DNA repair response, increases phosphorylated KAP1, resulting in transcriptional activation of XIST, the lncRNA that inactivates the X chromosome. We elucidate whether the increase in XIST competitively recruits the deacetylase SPEN away from HERV-K, resulting in an increase in HERV-K expression and consequent viral innate immune interferon (IFN)-STAT1 signaling. We determine whether this is a feature of MON in all forms of PAH, particularly in females, and persists in PAH MO-DC and MO-MØ. We investigate whether the propensity to apoptosis in PAH MON is related to IFN-STAT1 signaling and results in differentiation of surviving MON that invade pulmonary arteries to become pro-inflammatory MO-DC and MO-MØ. In Aim 2, we add MON differentiated from induced pluripotent stem cells (iPSC) (iMON) to novel bio-fabricated vascular tubes perfused under physiologic or high shear stress and populated with iPSC-differentiated endothelial cells (iEC) lining the lumen surrounded by circumferentially arranged iPSC-differentiated smooth muscle cells (iSMC) derived from PAH patients carrying a BMPR2 mutation or from control subjects. Single cell RNA Seq is applied to find mechanisms explaining (i) differentiation of iMON to iMO-DC and iMO-MØ, (ii) iMO-DC induced iSMC proliferation, and (iii) iMO-MØ impaired phagocytosis. Monocytes from athymic rats treated with SUGEN 5416 where females develop more severe PAH than males, and Bmpr2+/- rats treated with 5-lipoxygenase that develop PAH with no gender bias will be assessed for the innate immune response observed in human PAH MON. Two strategies will be used to trace MON differentiation to MO-DC and MO-MØ during the evolution of PAH in the rats and we will determine whether blocking IFN prevents pulmonary vascular remodeling and PAH. In Aim 3 CRISPR inhibitory technology is applied in which iPSC expressing dCAS9KRAB are differentiated to iMON and transduced with guides targeting a reduction in 9 genes expressed in MON and mutant in PAH. We will determine whether these cells share a common pathway of gene dysregulation. Bio-fabricated vascular tubes populated with iEC and iSMC with a reduction in these PAH genes are used to determine the contribution of mutant vascular cells to iMON differentiation to iMO-DC and iMO-MØ. Genome wide CRISPR screens are applied to find regulators of HERV-K expression and agents that improve PAH MØ phagocytosis. By uncovering the mechanism causing...