(325h) Matrix Stiffness Regulates Proteome Profiling of Primary Hepatocytes and Extracellular Vesicles Secretion upon Alcohol Exposure and HIV Infection

Background: Chronic liver diseases affects over 75 million people worldwide. The major cause of liver disease includes excessive consumption of alcohol and viral infections. Alcohol contributes to a more severe liver disease progression specifically for HIV infected patients resulting in hepatocytes cell death and impacted extracellular vehicles (EVs) biogenesis. EVs secretion is a form of cell-cell communication by delivering cargoes which contain significant proteins that that are major contributions in liver diseases. Due to alcohol abuse in HIV-infected patients, it is unclear whether alcohol interferes with liver diseases progression in the viral infected patients. Liver stiffness (LS) is a clinically well accepted marker for fibrosis progression and is driven by alteration of extracellular matrix proteins in liver. Although LS is well established as a clinical indicator for liver diseases, the role of LS that potentiates the changes in hepatocytes physiology with alcoholic patients and metabolic dysfunction in HIV-infected patients is still under explored. We hypothesize that stiffness alters hepatocytes proteins expression and impacts liver biology in alcoholic and HIV-infected patients, hence exacerbate the disease progression via EVs secretion.

Materials and Methods: To address this gap in knowledge, we have engineered an innovative biomimetic stiffness platform “BEASTS (Bio-Engineered Adhesive Siloxane substrate with Tunable Stiffness)” that recreates physiologic (2 kPa) and pathologic stiffness (25 kPa). We performed proteomic analysis on primary human hepatocytes culture on 2kPa and 25kPa on all upon alcohol exposure and HIV infection on all the four treatments (control, EtOH, HIV, EtOH-HIV) on soft and stiff microenvironments; The EVs that were isolated from these conditions were also quantified. We generated heatmaps and PCA analysis, performed biological process analysis, disease enrichment, and other bioinformatic analysis for all the conditions.

Results and Conclusion: We found several significant proteins that are differentially expressed on both soft and stiff substrate. Several proteins link to EtOH metabolism and HIV infections are moderately expressed in EtOH and HIV-infected hepatocytes on soft microenvironments but highly expressed on the stiff conditions. We found a clear distinct correlations of proteins expression on all conditions when performing PCA. We further confirm that stiffness plays a plausible role in both alcoholic liver disease and viral infected patient. Targeting those highly expressed proteins might be potential treatments for chronic liver diseases.