The Cellular Response to Elafin in PAH

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Project 1 builds upon exciting preclinical studies showing efficacy of daily subcutaneous administration of an elastase inhibitor, human recombinant Elafin, in reversing a severe model of pulmonary hypertension in rats. Elafin also induces significant regression of neointimal lesions in pulmonary arteries (PA) in explants from lungs of patients with pulmonary arterial hypertension (PAH), and unexpectedly improved deficient BMPR2 signaling in endothelial cells (EC) from patients with PAH by recruiting BMPR2 receptors to caveolae. Moreover, the elevated circulating levels of neutrophil elastase that Elafin would be expected to inhibit and the favorable toxicity and pharmacokinetic profile of Elafin, have led us to address the biology of Elafin, in a manner, we hypothesize, that will attribute Elafin efficacy in PAH to its properties of subverting the adverse functions of neutrophils (PMN) and of reversing vascular cell dysfunction. In Aim 1, we will investigate the role of Elafin in controlling abnormal PAH PMN function and PAH PMN-PAEC interactions under static conditions, of flow as well as laminar and disturbed flow. To investigate the mechanisms involved we will use a candidate approach, as well as RNA Seq and assays of transposase accessible chromatin (ATAC Seq), applicable to small numbers of PA EC under conditions of static, laminar and disturbed flow. In Aim 2, we will determine whether promising preliminary studies that show an Elafin-responsive phenotype in native PAEC that is reproduced in iPSC-EC from the same patients is true for a larger cohort. We will then relate the biologic improvement with Elafin to a gene expression signature. We will generate iPSC-EC from patients in the Phase 1 and 2 Clinical Trials in Project 3, and correlate biologic measurements of the iPSC-EC response to Elafin with gene expression and with clinical outcome as this relates to Elafin responders vs. non-responders. In Aim 3 we will cast a wider net for Elafin function in cells as we delineate the Elafin interactome in PMN and PAEC. This will be accomplished by immunoprecipitation (IP) with labeled Elafin and mass spectrometry to identify the interacting proteins. Once validated by co-IP, the impact of Elafin has on the function of the interacting protein will be assessed. In Aim 4, we will build upon compelling preliminary data showing the impact of a novel mass spectrometry-flow cytometry hybrid technology (CyTOF) in identifying a pan-PAH signature that suggests activation of CD4+CD25hi cells, and will determine the extent to this and other abnormalities can be normalized ex vivo by Elafin both acutely and in patients with PAH following chronic treatment. We will relate CyTOF to the biology and the state of activation of cells within the perivascular niche, using multiplex ion beam imaging (MIBI). The studies proposed allow an unprecedented opportunity to advance knowledge in the broader fields of PMN-EC interaction, flow dependent EC gene and epigenetic regulation, and correlation of PBMC activation with changes in the perivascular niche. We will also be uniquely positioned to identify and predict a positive response to Elafin, and alternative strategies for the non-responder.