Abstract 4143098: Development and characterization of an ex vivo human precision-cut lung slice (hPCLS) system for target validation for pulmonary arterial hypertension

Background: Translating therapeutic target biology from in vitro culture systems and animal models to humans remains a significant challenge in drug discovery. Recently, human precision-cut lung slices (hPCLS) have emerged as an innovative ex vivo model to better understand the pathophysiology of pulmonary disease. Given the difficulty in acquiring lung tissue from patients with pulmonary arterial hypertension (PAH), we aimed to develop a hPCLS culture system from healthy donors that replicates the PAH phenotype. Methods: Cryopreserved hPCLS from several healthy donors were obtained from AnaBios. Viability of the hPCLS was assessed using the lactate dehydrogenase (LDH) assay on cultured media. The hPCLS were cultured for seven days with growth factor (GF) cocktails containing various combinations of EGF, FGF, endothelin-1 (ET-1), PDGF-β, activin A, and TGF-β for 7 days to induce PAH-like phenotypes. The stimulated hPCLS were analyzed using RNA sequencing (RNA-seq). Histological analysis assessed pulmonary arterial (PA) medial wall thickness. RNA-seq data from human lungs with and without PAH served as a reference. Gene expression patterns were compared across key disease-associated pathways and processes. The effects of α5β1 integrin inhibition were investigated using the antibody M200 in the PAH-like hPCLS system. Results: LDH activity remained low across all treatment groups over the seven-day period, indicating no tissue toxicity. Histological analysis with H&E and EVG staining showed increased medial wall thickness and elastin deposition in pulmonary arteries stimulated by GFs. RNA-seq analyses revealed gene expression changes in response to several GFs that were consistent with the reference PAH dataset. Key gene sets related to extracellular matrix formation, integrin signaling, proliferation, senescence, and growth factor signaling showed similarities between several GFs and human PAH. Moreover, treatment with the α5β1 antibody, M200, in GF-stimulated hPCLS demonstrated modulation of PAH-like phenotypes. Conclusions: We successfully established a novel ex vivo model using hPCLS from healthy donors that mimics PAH characteristics when stimulated by GFs (ET-1, FGF, PDGF-β, and EGF). This model also demonstrated that α5β1 inhibition reduced PAH-like phenotypes. This hPCLS model, in conjunction with tissue from PAH patients, could be a valuable tool for evaluating new therapies, enhancing the translation from preclinical studies to human applications.