Novel Morphological Profiling Assay Connects ex Vivo Endothelial Cell Responses to Disease Severity in Liver Cirrhosis

Abstract

Endothelial cell (EC) dysfunction in response to circulating plasma factors is a known causal factor in many systemic diseases. However, no appropriate assay is available to investigate this causality exvivo. In liver cirrhosis, systemic inflammation is identified as central mechanism in progression from compensated to decompensated cirrhosis (DC), but the role of ECs therein is unknown. We aimed to develop a novel exvivo assay for assessing EC responses to patient-derived plasma (PDP) and assess the potential of this assay in a cohort of liver cirrhosis patients. Image-based morphological profiling was utilized to assess the impact of PDP on cultured ECs. Endothelial cell (EC) monolayers were exposed to 25% stabilized PDP (20 compensated cirrhoses, 20 DCs, and 20 healthy controls (HCs). Single-cell morphological profiles were extracted by automated image-analysis following staining of multiple cellular components and high-content imaging. Patient profiles were created by dimension reduction and cell-to-patient data aggregation, followed by multivariate-analysis to stratify patients and identify discriminating features. Patient-derived plasma (PDP) exposure induced profound changes in EC morphology, displaying clear differences between controls and DC patients. Compensated cirrhosis patients showed overlap with healthy controls and DC patients. Supervised analysis showed Child-Pugh (CP) classcould be predicted from EC morphology. Most importantly, CP-C patients displayed distinct EC phenotypes, in whichmitochondrial changes were most discriminative. Morphological profiling presents a viable tool to assess the endothelium exvivo. We demonstrated that the EC phenotype corresponds with disease severity in liver cirrhosis. Moreover, our results suggest the presence of mitochondrial dysfunction in ECs of CP-C patient.