Current Pilot Project Awardees

Project Title: Rewiring Pathways in Liver Metabolism

Liver disease has become a significant health concern worldwide with conditions such as obesity and liver cancer on the rise. Dysregulation of de novo lipogenesis (DNL) is a large factor in these diseases as it is the metabolic pathway responsible for the production of the majority of lipids in the liver. Knowing the rate of DNL in an individual may benefit clinicians as it would provide early warnings of the development of these diseases and provide a way to assess treatments. Therefore, it is necessary to study DNL at the cellular level, including how responses to external stimuli such as competing pathways mediate overall metabolic rates. My research group has recently imaged DNL of living hepatoma cells at super-resolution in the infrared by optical photothermal infrared microscopy, allowing us to visualize the spatial and temporal heterogeneity of DNL at the sub-cellular level. Here we extend our studies to real-time visualization and quantification of hepatocyte metabolism and its adaptation to environmental stress under nearly label-free conditions in living cells. Our platform will transform our ability to study DNL and, coupled with biochemical and biophysical tools, manipulate metabolic activity. Unraveling these interactions will improve diagnoses and therapies that target metabolic networks in metabolic disease.

Project Title: Mechanisms of vascular Dysfunction in Cystic Fibrosis-related Liver Disease

Cystic fibrosis-associated liver disease (CFLD) affects up to 30% of patients with cystic fibrosis (CF). A severe form of portal hypertension in the absence of cirrhosis, characterized by obliterative portal venopathy, has recently been described in CF patients undergoing liver transplantation. Recent work has shown that endothelial cells do express CFTR, however, the pathogenetic mechanism of non-cirrhotic portal hypertension in CF is unknown. Moreover, our preliminary data show that CFLD is linked to intestinal dysbiosis. The hypothesis of this project is that vascular dysfunction in CFLD results from an increased inflammatory reaction to gut-derived bacterial products in the endothelial cells of the portal vein radicles. This project aims at understanding the mechanisms leading to the portal vascular dysfunction in CF and how defective CFTR function affects the physiology of the endothelium. Single cell transcriptomics will be used to characterize the portal inflammation and endothelial damage in CFTR-defective mice. In vivo treatments will be used to evaluate how modulation of the gut microbiota and intestinal permeability could prevent the vascular damage. We will also use human induced pluripotent stem cells-derived endothelial and biliary cells to understand the function of CFTR in the endothelium and the relationship between endothelial cells and the biliary epithelium. This project is designed to unveil the presence of pathogenetic cross talk mechanisms between biliary and endothelial cells that are activated in response to gut-derived factors and possibly causing liver vascular disease in patients with CF.

Project Title: Ex Vivo Liver Perfusion for Nanoparticle Delivery to Sinusoidal Endothelial Cells

In 2021, over 13,000 people were added to Liver Transplant Waitlist, the largest single year addition. With only 9,500 livers recovered, there is a need to transplant as many livers as possible without risking patient health and safety. Furthermore, around 10% of the recovered livers were declined for transplant, further reducing the already limited supply. Therefore, it is critical to improve the function of as many marginal organs as possible. One issue that is exacerbated in marginal organs is ischemia reperfusion injury (IRI). Current studies have implicated that nitric oxide, derived from endothelial nitric oxide synthase, can have a protective benefit during IRI. In marginal livers, the sinusoidal endothelial cells may have also undergone capillarization. This disease state precedes fibrosis and can result from many different etiologies. A GTPase of the immunity-associated protein family member 5 (GIMAP5) has been shown to be critical in the transition from healthy to capillarized endothelial cells. We hypothesize that it would be useful to exclusively target endothelial cells in the liver while avoiding other cell types to prevent off target effects and toxicities. Ex vivo perfusion (EVP) gives us a unique opportunity to assess targeting exclusively in the liver and is a clinically relevant transplant application. The goal of this proposal is to address IRI and capillarization in endothelial cells during liver EVP using antibody-targeted nanoparticles to deliver nucleic acids.

Project Title: Fractalkine signaling in liver sinusoidal endothelial cells

In response to tissue infection or injury, circulating leukocytes migrate across the endothelial layers to reach the tissue to eliminate pathogens, promote wound healing, or regulate local inflammatory responses. Transendothelial migration occurs in two paths, either at the junction between endothelial cells (paracellular) or through endothelial cells (transcellular). Comparing to paracellular migration, which involves the opening of tight-junctions, transcellular migration was considered to better maintain the integrity of endothelial layer although it remains unclear how endothelial cells remodel their membrane and cytoskeleton to create tunnels to accommodate the penetration of leukocytes. Liver sinusoidal endothelial cells (LSECs) present a preferred model for investigating transcellular migration because it was observed at high frequency in LSECs. Importantly, LSECs play a critical role in regulating the number and activation of infiltrating leukocytes although the underlying mechanism is poorly understood. In this proposal, we aim to delineate the signaling pathway that promotes transcellular migration in LSECs. Biochemical reconstitution, high-resolution microscopy, together will cell engineering approaches will be implemented. This work is expected to identify a novel receptor, FKN, that promotes transcellular migration through LSECs. It will provide molecular insights into understanding the causes of inflammation-related liver injury.

Project Title: Understanding the Type I Interferon Response to Amanitin Poisoning

Acute poisonings are a significant cause of mortality and morbidity worldwide, yet modern treatment options are lacking and often limited to supportive care. A prototypical, increasingly common cause of poisoning is amanitin poisoning, which is caused by accidental ingestion of mushrooms of the amanitin genus. Amanitin poison causes fulminant liver and kidney injury, and current therapies are all limited to supportive care and organ transplantation. Whether our immune system, which protects us from pathogenic infection and wounding, also protects us from poisoning is an unknown fundamental question in biology. Additionally, the logic underlying how cells sense poisons, if tailored immune responses to different classes of poisons exist, and how these classes would be organized, remains undefined. To explore this, we selected 24 natural poisons mice in natural environments may accidentally ingest while foraging for food that are simultaneously relevant to human health and disease. These poisons included fungal-derived poisons like amanitin, plant-derived poisons like triptolide and colchicine, and soil bacteria-derived poisons like oligomycin. Mice were poisoned with a single dose based on exposures quantities described in humans, and RNA sequencing was performed on the liver, the major detoxifying organ, of poisoned mice four hours post-poisoning in order to capture the early transcriptional response. Principle components analyses revealed class-specific anti-poison immunologic transcriptional responses, which surprisingly were organized based on the cellular process known to be disrupted by the poison. We discovered a specific immune response against aminitin, a frequent cause of human poisoning leading to intensive care hospitalization, and other chemically distinct transcriptional inhibitors for which there are no therapies beyond supportive care and liver transplantation. Therapeutic manipulation of this immune response greatly reduced liver damage and mortality in animal models. This project seeks to dissect the mechanisms of immune activation by transcriptional poisons like aminitin and how it contributes to host protective host defense that can become pathophysiologic and drive fatal liver disease.