Pulmonary Artery Endothelial Cell Endotypes and the Role of Anoikis Resistance in Pulmonary Hypertension

Abstract Pulmonary arterial hypertension (PAH) is a devastating disease marked by endothelial cell (EC) dysfunction. While EC dysfunction is at the core of PAH pathobiology, EC phenotypes are incompletely characterized and remain controversial. Current methods to study this gap source cells from end-stage patients, non-diseased cell lines, or outside of the pulmonary vasculature. Right heart catheterization is the fundamental diagnostic procedure in PAH and is repeated throughout the disease course. We have shown that ECs from the balloons of pulmonary artery catheters can be harvested, propagated ex vivo and characterized to elucidate molecular mechanisms of EC dysfunction. We contend that ECs in PAH display abnormal programmed cell death triggered by cell detachment from the vessel wall, known as anoikis resistance. This proposal will leverage our “cell biopsy” method to define EC dysfunction in human PAH and patient, time and biomechanical factors that influence EC endophenotypes. Our central hypothesis is that anoikis resistance is a central paradigm in progressive PAH and that ECs from patients with advanced PAH will demonstrate dysregulated anoikis as compared to those with less severe disease and other pulmonary hypertension subtypes. We will determine the anoikis mediators (e.g., syndecan-4, and PLAC8) that promote this pathophenotype and the extent to which targeting them can reverse anoikis resistance. We will characterize sample microheterogeneity with single-cell sequencing of primary ECs and singe-cell cloning of propagated cells to study anoikis resistant subpopulations and their adhesive properties. Transcriptomics will be integrated in silico to identify additional therapies via a drug repositioning strategy to rescue anoikis resistance in vitro. Our experiments will include ECs from patients with different forms of pulmonary vascular disease, diseased controls, and biological replicates from the same patients over time. We will compare the signatures from our more central pulmonary artery ECs to that of established microvascular PAH ECs. Together, these aims will inform new mechanistic targets to reverse anoikis resistance in PAH as well as a personalized drug screening approach over the course of pulmonary vascular disease.