Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD) is the most prevalent form of chronic liver disease worldwide. The disease is closely associated with dyslipidemia; however, the limited understanding of its molecular mechanisms hinders accurate prediction of disease progression and the development of effective diagnostic and therapeutic approaches. Dyslipidemia is also observed in COVID-19 patients, and its severity correlates with disease progression and outcome. Given the significant healthcare and economic burden of MASLD and COVID-19, the search for new biomarkers for both diseases is of utmost importance. The aim of the dissertation was to identify potential tissue and blood biomarkers for different stages of MASLD fibrosis and for the severity and progression of COVID-19. Additionally, we investigated how impaired cholesterol biosynthesis affects membrane sterol composition and the binding of the SARS-CoV-2 virus to host cell membranes.
Through transcriptome analysis of 60 liver samples from patients at different fibrosis stages, we identified nine differentially expressed genes (AEBP1, ANKRD29, CPE, EFEMP1, ITGBL1, LUM, PTGDS, A2M, TRIM22) across various groups. Based on additional computational analyses and a review of the published literature, we expanded the set of target genes and experimentally validated the differential expression of 17 of the 27 genes in a larger patient cohort (N=141) using RT-qPCR. In addition, using machine learning techniques, we developed a classification model trained on three clinical parameters (body mass index, total cholesterol, and platelet count) and the expression of eight genes (LUM, PTGDS, FBLN5, DPT, EFEMP1, STMN2, DCN, MMP2) to stratify patients with early fibrosis stages (F0–F1/F2; AUC=0.84).
Using targeted lipidomics, we determined the sterol profile in the serum of 62 hospitalized COVID-19 patients and provided insights into sterol intermediates, the precursors of cholesterol, during the course of COVID-19. Our findings indicate that cholesterol biosynthesis is altered during COVID-19. Compared to patients with a milder disease course, those with severe COVID-19 exhibited more pronounced changes, with statistically significant alterations in the concentrations of several sterol intermediates (zymostenol, zymosterol, 24-dehydrolathosterol, desmosterol and cholesterol). Using machine learning techniques, we have developed a classification system (N=164) that outperforms existing clinical risk assessment tools. Our model is based on eight readily accessible clinical parameters and predicts COVID-19 disease progression with exceptional accuracy (AUC=0.96). Adding measurements of a subset of sterol intermediates improves the sensitivity of the model.
We also investigated the effect of impaired cholesterol biosynthesis on membrane sterol composition and spike protein binding to membranes using HepG2 model cell lines with knockout genes involved in the post-squalene part of cholesterol biosynthesis. Using LC-MS/MS and confocal microscopy, we demonstrated that impaired cholesterol biosynthesis alters the sterol composition of the investigated cells and affects the specific binding of the fluorescent variant of ostreolysin A6 to lipid rafts. To investigate the binding of the viral spike, we successfully expressed and purified the receptor-binding domain (RBD) of the spike protein using recombinant DNA techniques and affinity chromatography. Our results suggest that ACE2 receptor expression varies among HepG2 cell lines of different genotypes. However, we were unable to confirm the effect of impaired cholesterol biosynthesis on the binding of RBD spike protein to HepG2 cell membranes of different genotypes.
The study identified novel potential tissue biomarkers for different stages of MASLD fibrosis and provided a basis for the discovery of non-invasive blood biomarkers for the disease. Additionaly, changes in the serum sterol profile of hospitalized COVID-19 patients were characterized and biomarkers associated with COVID-19 progression were identified. In addition, guidelines for future studies on the effects of impaired cholesterol biosynthesis on the binding and entry of the SARS-CoV-2 virus into host cells were established.
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