Metabolic fibrotic profiling during in vitro activation of primary mouse hepatic stellate cells

Background: Liver fibrosis is characterized by the excessive buildup of extracellular matrix (ECM) proteins. Among the various cell types present in the liver, hepatic stellate cells (HSCs) are primarily responsible for regulating ECM balance. When liver injury occurs, damaged hepatocytes send signals to stellate cells, triggering their activation and resulting in the formation of scar tissue. While the pathophysiology of the disease process is well understood, the cellular metabolism underlying the HSC activation process remains largely unclear. During this activation process, HSCs adjust to the increased energy demands by altering various metabolic pathways.
Aim and methods: To determine the fibrotic metabolic profile during stellate cell activation through high-resolution mass spectrometry (HRMS) based metabolomics. We cultured freshly isolated mouse HSCs in regular culture dishes which induces a rapid HSC activation partly mimicking the in vivo HSC activation. We confirmed their activation at day 0, 1, 4 and 7 in culture by immunostaining for alpha SMA and Desmin, mRNA analysis of activation markers Acta2, Col1a1 and Lox and determined their gene expression profile by QuantSeq RNA sequencing. The metabolic profile of the HSCs was performed by employing Liquid-Liquid extraction with 80% Methanol, followed by Hydrophilic interaction liquid chromatographic based separation with ACQUITY UPLC BEH Amide Column and analysed on Vion IMS QTof mass spectrometric platform.
Results and conclusions: We optimized primary HSC cell numbers, extraction solvent, chromatographic and MS parameters. Our study revealed amino acid perturbations at different time points during in vitro HSC activation process. These perturbations are reflected in many pathways which are related to liver fibrosis such as Inflammation, oxidative stress, ECM remodelling, energy pathway metabolism, cholesterol & fatty acid metabolism and glycogen & galactose metabolism. The metabolomics data will be integrated with the RNA sequencing data and the HRMS method developed will be expanded to examine the global profile of metabolic changes to integrate with the RNA sequencing profiles obtained. Thus, our preliminary results underscore the importance of studying amino acids in the context of hepatic stellate cell activation to advance our understanding of liver fibrosis, a common pathological feature in chronic liver diseases.