Pulmonary arterial hypertension (PAH) is a rare, but progressive and fatal disease that is characterized by diffuse remodeling of the pulmonary vascular bed, the mechanisms responsible for these diffuse changes are poorly understood. Numerous studies have examined whole lung gene expression to better understand PAH pathogenesis. Altered expression in genes that regulate metabolism, growth, and inflammation have been demonstrated, however the field has been greatly limited by the inability to evaluate temporal, cell-specific changes in gene expression. The long-term goal of this proposal is to employ recently developed image-based spatial transcriptomic techniques (spatial profiling) to identify gene expression profiles that are unique to specific cell types and only those cells that are involved in the pathologic lesions seen in PAH. Successful completion of this project will significantly advance our understanding of the pathobiology of PAH and will identify the most important cellular pathways to target for novel therapeutics to treat PAH. The specific aims of this proposal are: 1) Use spatial profiling to determine how PAH impacts specific cell types in situ. 2) Establish in vitro cellular models of PAH by using single cell RNA sequencing (scRNAseq) to identify and isolate pulmonary vascular cells from whole lung tissue that match the genetic profile of the pathologic cells obtained in Aim 1. In Aim 1, Spatial profiling will be used to determine cell specific changes in gene expression in lungs from rats using the sugen/hypoxia model of PAH. In Aim 2, cells will be isolated from the lungs in this rat model and compared to the spatial transcriptomics obtained from Aim 1 by using scRNAseq. This will allow identification of markers to isolate in culture the specific cells that most closely model the disease, thereby providing an invaluable new technique for in vitro study of the pathologic cells responsible for pulmonary vascular remodeling in PAH. This project leverages state-of-the-art novel technologies to identify cell-specific changes in gene expression and to develop an innovative in vitro model to study cellular abnormalities in PAH. Ultimately, this work will provide critical insights into the relationship among the cells in the lung that leads to PAH, generate breakthrough data to redefine the field, reveal new therapeutic opportunities to combat PAH and lay the foundation to earn federal funding to pursue these innovate studies.