Endothelial Injury, BMPR2 Dysfunction and Macrophage Activation Cause EndMT and PAH

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A considerable body of knowledge indicating that inflammation and immune dysregulation play a pivotal role in the development of pulmonary arterial hypertension (PAH) have now led to the first immunotherapy trials for this condition. However the success of this approach requires that we determine how the aberrant immune/inflammatory response to pulmonary artery endothelial cell (PAEC) injury develops and contributes to the evolution of the occlusive arteriopathy, that is characterized by apoptosis, apoptosis resistance and endothelial mesenchymal transition (EndMT). It is also important to relate these features to dysfunction of a key genetic modifier of PAH, bone morphogenetic protein receptor (BMPR)2. This proposal brings unique focus to two key innate immune mediators that are highly expressed in perivascular macrophages (MØs) from patients with PAH and that mount a coordinated attack on the pulmonary vasculature: the leukotriene, LTB4, and the endogenous retrovirus, HERV-K, an increasingly recognized cause of autoimmune injury. The overall hypothesis is injured PAECs with dysfunctional BMPR2 signaling recruit and activate MØs that amplify LTB4 and HERV-K; these immune factors work in concert to sustain inflammation and promote severe PAH by apoptosis and EndMT. Aim 1 evaluates how LTB4 may be autonomously produced by injured PAECs, which in turn activate MØs to stimulate further LTB4 biosynthesis to cause PAEC apoptosis and EndMT when BMPR2 signaling is impaired. This aim also explores how amplification of endogenous HERV-K may further stimulate LTB4 biosynthesis and how LTB4 can induce HERV-K expression in monocytes. Single cell RNA Seq will genotype PAEC populations that are fated for apoptosis or EndMT in response to LTB4 to delineate gene expression changes that mark the transition to apoptosis resistance and EndMT. Aim 2 evaluates whether PAECs from PAH patients including those with reduced BMPR2 function, secrete factors in response to oxidant or inflammatory injury, that amplify HERV-K expression in monocytes. This aim also considers the additive effects of loss of BMPR2 in monocytes, on amplification of HERV-K and whether transcriptional and translational mechanisms are involved in this process. Also to be investigated is whether the secreted HERV-K product, dUTPase, promotes EndMT in PAEC with BMPR2 dysfunction that are primed to undergo transformation. Single cell RNA Seq analyses will elucidate gene expression changes induced by HERV-K in the PAEC evolution to EndMT. Aim 3 investigates the vulnerability of BMPR2-deficient animals to develop severe PH and EndMT using a mouse with loss of BMPR2 in fate mapped EC, and a new transgenic rat PH model with BMPR2 haploinsufficiency. The animals are treated with endotracheal instillation of Ad-5LO to generate high LTB4 levels or recombinant HERV-K dUTPase given intravenously. Strategies to reverse EndMT and severe PH will be tested that have translational relevance to the fatal obliterative vasculopathy in PAH patients.