Biomimetic Approach to Elucidate the Role of Microenvironment in Regulating Cell Communications in Liver Diseases

Liver is a complex organ but often underappreciated for its functions to remove toxins and fight infection in the body. Despite the ongoing prevalence of patients contracting chronic liver diseases, coupled with a global healthcare investment of approximately $70 billion, there is still no effective treatment for chronic liver diseases including alcoholic liver disease. The purpose of this dissertation was to understand the interactions of parenchymal and non-parenchymal liver cells in physiological and pathological like environments. This allowed us to recreate a healthy and the diseased microenvironments to observe the stages of the liver diseases development and apply appropriate treatments for the liver diseases. Substrate stiffness was used as a biomimetic approach to study the liver cells function and observe the differences of liver cells behavior when cultured on different microenvironments. Cell-cell communication, proteomic approach, and a novel drug delivery system were investigated to provide a potential treatment to chronic related liver diseases such as alcoholic liver disease. Hepatic stellate cells were found to enhance their functions in co-culture compared to a mono-culture system. EVs isolated from hepatocytes cultured on soft and stiff substrate revealed significant proteins involved in disease developments and were found to potentially serve as biomarkers for the early diagnosis for liver diseases. Treating with an antioxidant drug, vitamin E, the oxidative stress was found alleviated. Together, stiffness shows a plausible role in developing liver diseases and EVs isolated from hepatocytes cultured on various stiffnesses as well as lipid nanoparticles encapsulated with vitamin E should also be considered for the future treatment of alcoholic related liver diseases

Role of Liver Stiffness and Alcohol on HBV Infection Pathogenesis

Hepatitis B Virus (HBV) is an infection that specifically targets hepatocytes and persistence of infection leads to inflammation and liver injury. The metabolism of alcohol is also known to cause injury and inflammation in the liver. The extent of liver damage can be analyzed by determining the pressure of the tissue with an ultrasound. As you go from a healthy liver to a fibrotic liver, the pressure increases from around 2 kPa to greater than 12.5 kPa. Previous studies have found that liver stiffness affects the primary hepatocyte function and cell interaction, but the exact mechanism behind the combined role of liver stiffness and alcohol in HBV infection is still unclear. This study aimed to determine the effect of liver stiffness and alcohol metabolism on HBV infection pathogenesis. To accomplish this aim, we used a soft and stiff liver model engineered specifically to a 2 kPa (healthy liver tissue) and 25 kPa pressure (fibrotic liver tissue). HBV transfected HepG2.2.15 cells were plated on these liver model plates. To mimic alcohol metabolism, the cells were exposed to Acetaldehyde Generating System (AGS). Results showed that liver stiffness significantly increased HBV infection markers and decreased the interferon alpha signaling by up regulating USP-18. In addition, liver stiffness increased inflammasome and pro-fibrotic markers in HBV transfected cells. The combination of alcohol metabolism with liver stiffness potentiated the HBV infection. We conclude that liver stiffness impairs interferon alpha signaling thereby increasing HBV persistence, which leads to liver inflammation and fibrosis. This study of the liver environment’s role in HBV infection and alcohol metabolism paves the way to new treatment options for patients as well as introduces more accurate lab models for research.https://digitalcommons.unmc.edu/surp2021/1023/thumbnail.jp

Increased liver stiffness promotes hepatitis B progression by impairing innate immunity in CCl4-induced fibrotic HBV\u3csup\u3e+\u3c/sup\u3e transgenic mice

Background: Hepatitis B virus (HBV) infection develops as an acute or chronic liver disease, which progresses from steatosis, hepatitis, and fibrosis to end-stage liver diseases such as cirrhosis and hepatocellular carcinoma (HCC). An increased stromal stiffness accompanies fibrosis in chronic liver diseases and is considered a strong predictor for disease progression. The goal of this study was to establish the mechanisms by which enhanced liver stiffness regulates HBV infectivity in the fibrotic liver tissue. Methods: For in vitro studies, HBV-transfected HepG2.2.15 cells were cultured on polydimethylsiloxane gels coated by polyelectrolyte multilayer films of 2 kPa (soft) or 24 kPa (stiff) rigidity mimicking the stiffness of the healthy or fibrotic liver. For in vivo studies, hepatic fibrosis was induced in C57Bl/6 parental and HBV+ transgenic (HBVTg) mice by injecting CCl4 twice a week for 6 weeks. Results: We found higher levels of HBV markers in stiff gel-attached hepatocytes accompanied by up-regulated OPN content in cell supernatants as well as suppression of anti-viral interferon-stimulated genes (ISGs). This indicates that pre-requisite “fibrotic” stiffness increases osteopontin (OPN) content and releases and suppresses anti-viral innate immunity, causing a subsequent rise in HBV markers expression in hepatocytes. In vitro results were corroborated by data from HBVTg mice administered CCl4 (HBVTg CCl4). These mice showed higher HBV RNA, DNA, HBV core antigen (HBcAg), and HBV surface antigen (HBsAg) levels after liver fibrosis induction as judged by a rise in Col1a1, SMA, MMPs, and TIMPs mRNAs and by increased liver stiffness. Importantly, CCl4-induced the pro-fibrotic activation of liver cells, and liver stiffness was higher in HBVTg mice compared with control mice. Elevation of HBV markers and OPN levels corresponded to decreased ISG activation in HBVTg CCl4 mice vs HBVTg control mice. Conclusion: Based on our data, we conclude that liver stiffness enhances OPN levels to limit anti-viral ISG activation in hepatocytes and promote an increase in HBV infectivity, thereby contributing to end-stage liver disease progression

Increased liver stiffness promotes hepatitis B progression by impairing innate immunity in CCl4-induced fibrotic HBV+ transgenic mice

BackgroundHepatitis B virus (HBV) infection develops as an acute or chronic liver disease, which progresses from steatosis, hepatitis, and fibrosis to end-stage liver diseases such as cirrhosis and hepatocellular carcinoma (HCC). An increased stromal stiffness accompanies fibrosis in chronic liver diseases and is considered a strong predictor for disease progression. The goal of this study was to establish the mechanisms by which enhanced liver stiffness regulates HBV infectivity in the fibrotic liver tissue.MethodsFor in vitro studies, HBV-transfected HepG2.2.15 cells were cultured on polydimethylsiloxane gels coated by polyelectrolyte multilayer films of 2 kPa (soft) or 24 kPa (stiff) rigidity mimicking the stiffness of the healthy or fibrotic liver. For in vivo studies, hepatic fibrosis was induced in C57Bl/6 parental and HBV+ transgenic (HBVTg) mice by injecting CCl4 twice a week for 6 weeks.ResultsWe found higher levels of HBV markers in stiff gel-attached hepatocytes accompanied by up-regulated OPN content in cell supernatants as well as suppression of anti-viral interferon-stimulated genes (ISGs). This indicates that pre-requisite “fibrotic” stiffness increases osteopontin (OPN) content and releases and suppresses anti-viral innate immunity, causing a subsequent rise in HBV markers expression in hepatocytes. In vitro results were corroborated by data from HBVTg mice administered CCl4 (HBVTg CCl4). These mice showed higher HBV RNA, DNA, HBV core antigen (HBcAg), and HBV surface antigen (HBsAg) levels after liver fibrosis induction as judged by a rise in Col1a1, SMA, MMPs, and TIMPs mRNAs and by increased liver stiffness. Importantly, CCl4-induced the pro-fibrotic activation of liver cells, and liver stiffness was higher in HBVTg mice compared with control mice. Elevation of HBV markers and OPN levels corresponded to decreased ISG activation in HBVTg CCl4 mice vs HBVTg control mice.ConclusionBased on our data, we conclude that liver stiffness enhances OPN levels to limit anti-viral ISG activation in hepatocytes and promote an increase in HBV infectivity, thereby contributing to end-stage liver disease progression

Biomimetic Approach to Elucidate the Role of Microenvironment in Regulating Cell Communications in Liver Diseases

Liver is a complex organ but often underappreciated for its functions to remove toxins and fight infection in the body. Despite the ongoing prevalence of patients contracting chronic liver diseases, coupled with a global healthcare investment of approximately $70 billion, there is still no effective treatment for chronic liver diseases including alcoholic liver disease. The purpose of this dissertation was to understand the interactions of parenchymal and non-parenchymal liver cells in physiological and pathological like environments. This allowed us to recreate a healthy and the diseased microenvironments to observe the stages of the liver diseases development and apply appropriate treatments for the liver diseases. Substrate stiffness was used as a biomimetic approach to study the liver cells function and observe the differences of liver cells behavior when cultured on different microenvironments. Cell-cell communication, proteomic approach, and a novel drug delivery system were investigated to provide a potential treatment to chronic related liver diseases such as alcoholic liver disease. Hepatic stellate cells were found to enhance their functions in co-culture compared to a mono-culture system. EVs isolated from hepatocytes cultured on soft and stiff substrate revealed significant proteins involved in disease developments and were found to potentially serve as biomarkers for the early diagnosis for liver diseases. Treating with an antioxidant drug, vitamin E, the oxidative stress was found alleviated. Together, stiffness shows a plausible role in developing liver diseases and EVs isolated from hepatocytes cultured on various stiffnesses as well as lipid nanoparticles encapsulated with vitamin E should also be considered for the future treatment of alcoholic related liver diseases

Exploring Interactions between Primary Hepatocytes and Non-Parenchymal Cells on Physiological and Pathological Liver Stiffness

Chronic liver disease is characterized by progressive hepatic fibrosis leading to the formation of cirrhosis irrespective of the etiology with no effective treatment currently available. Liver stiffness (LS) is currently the best clinical predictor of this fibrosis progression irrespective of the etiology. LS and hepatocytes-nonparenchymal cells (NPC) interactions are two variables known to be important in regulating hepatic function during liver fibrosis, but little is known about the interplay of these cues. Here, we use polydimethyl siloxane (PDMS) based substrates with tunable mechanical properties to study how cell–cell interaction and stiffness regulates hepatocytes function. Specifically, primary rat hepatocytes were cocultured with NIH-3T3 fibroblasts on soft (2 kPa) and stiff substrates that recreates physiologic (2 kPa) and cirrhotic liver stiffness (55 kPa). Urea synthesis by primary hepatocytes depended on the presence of fibroblast and was independent of the substrate stiffness. However, albumin synthesis and Cytochrome P450 enzyme activity increased in hepatocytes on soft substrates and when in coculture with a fibroblast. Western blot analysis of hepatic markers, E-cadherin, confirmed that hepatocytes on soft substrates in coculture promoted better maintenance of the hepatic phenotype. These findings indicate the role of stiffness in regulating the hepatocytes interactions with NPCs necessary for maintenance of hepatocytes function