(T0930-04-24) Identifying the Role of Prion-Like Protein Doppel as a Novel Mediator of Pulmonary Arterial Hypertension

Purpose: The severity of endothelial cell dysfunction and the underlying abnormalities in gene expression profiles vary among patients with pulmonary arterial hypertension (PAH). Identifying new biomarkers for PAH holds promise for improving personalized treatment responses and clinical outcomes. Here, we propose to identify novel markers of PAH and study their biology in the pathogenesis of PAH.

Methods: To identify new biomarkers, we fabricated a microfluidic chip capable of detecting and isolating circulating endothelial cells (CECs) directly from whole blood shed from the arteries affected by PAH. Subsequently, we conducted RNA sequencing on the isolated CECs (N = 4/group). Differentially expressed genes (DEGs) exhibiting an average four-fold difference between CECs collected from healthy individuals and those from PAH patients were assessed and PRND (Doppel) was selected as a top-tier candidate. We further explored the functional role of Doppel by genetically modifying PAH-ECs (source: PHBI) in vitro and in vivo using Sugen/hypoxia-induced Doppel knockout mice.

Results: The isolation of viable CECs followed by RNA sequencing analysis comparing healthy individuals to PAH patients unveiled 138 DEGs. Notably, genes upregulated or downregulated in PAH-affected CECs were implicated in PAH-specific signatures and angiogenic processes. Particularly, gene encoding a prion-like protein Doppel showed high upregulation in both PAH-CECs and PAH-ECs. Doppel overexpression in human PAH-ECs resulted in 1946 DEGs (padj≤0.05, -1.75≤fold change≤1.75); among them 15-17% DEGs matched with both DisGeNET and Mayaan PAH dataset. Hallmark Gene Set Enrichment Analysis of PAH-associated genes in our dataset strongly suggests the up-regulation of epithelial to mesenchymal transition (EMT) process. Doppel is known to regulate tumor angiogenesis by regulating VEGF-VEGFR2 axis but not in healthy vessels. We also noticed that Doppel protein is highly expressed in the isolated human PAH-ECs and in lung tissue of Sugen/hypoxia induced PAH model. Doppel blocking by its monoclonal antibody increased BMPR2 expression but decreased β-catenin, SNAIL/SLUG pathway and phospho-SMAD1/3/5, and thus, reversed the EMT in PAH-ECs. Doppel-knockout mice were protected from increasing pulmonary arterial pressure in SUGEN/hypoxia PAH model.

Conclusion: Our findings imply that the newfound marker Doppel is a crucial mediator in PAH progression.