Alcoholic liver disease (ALD) is one of the most common forms of chronic liver injury in the United States. Chronic ethanol consumption results in toxic metabolites in the liver and increases endothelial senescence and dysfunction leading to inflammation and liver damage. Aging is linked with the severity and poor prognosis of various liver diseases including alcoholic liver diseases and is associated gradual alteration of structure and function in liver tissues and cells including liver sinusoidal endothelial cells. Endothelial dysfunction is an early pathophysiological hallmark in the development of liver disease. Senescence, the cellular equivalent of aging, was proposed to be involved in endothelial dysfunction. Indeed, enhanced cellular senescence was associated with a poor outcome in alcohol-related fatty liver disease, while increased hepatic senescence mediators such as p21 and TNF-? that were related to liver/endothelial dysfunction in ALD cirrhosis. Increasing evidence supports that non-coding RNAs (ncRNAs) play a central role in various cellular pathways by regulating gene expression. Indeed, there is a strong link between ethanol metabolism and endotoxin/lipopolysaccharide (LPS) induced cellular senescence and endothelial dysfunction in ALDs. Hepatic sinusoids, connected directly to the portal circulation, serve as the first barrier against these inflammatory and noxious stimuli. We have novel preliminary data showing that selective ncRNA/microRNA genes are aberrantly expressed in liver specimens from ALD animals and are regulated and involved in ethanol metabolism. In particular, we have found: (a) intra-gastric ethanol feeding significantly increased the expression of cellular senescence initiators EGR1, PAI-1 and Id1 and silenced p53 effectors E2F1 and IGFBP3; (b) ethanol enhanced microRNA-34a and p53 expressions and altered their target genes such as SIRT1, PPAR? and HNF4?, which leads to senescence phenotypes in liver sinusoidal endothelial cells (LSECs); (c) Inhibition of miR-34a/p53/TLR4 by anti-miR-34a/p53 Morpholino and CRISPR/cas9 approaches has successfully recovered alcoholic liver injury in ethanol treated mice in vivo. Although the combined evidence supports a link between ncRNAs and alcoholic liver disease, there is a critical need to determine the underlying mechanism whereby ethanol-dependent miRNAs promote alcoholic liver injury. Our long-term goal is to determine underlying mechanisms contributing to alcohol- induced liver disease so that new mechanism-based, clinically effective prevention or treatment strategies can be developed. The objective for this proposal is to determine how ethanol- dependent ncRNAs mediate endothelial dysfunction and senescence in the progression of alcoholic liver diseases. Our central hypothesis is that ethanol-dependent miRNAs contribute to alcoholic liver injury through regulation of cellular senescence and dysfunction in endothelial cells. This hypothesis was formulated based upon the existing literature and our own preliminary data described above. The following three specific aims are proposed: First, we will determine the effects of ncRNA mediated cellular senescence and endothelial dysfunction in senescence accelerated mice with alcoholic liver injury in vivo (Aim 1). Second, we will identify the downstream targets of p53-miR-34a axis that are involved in endothelial senescence and dysfunction in miR-34a and p53 knockout mice in vivo (Aim 2). Further, we will determine the effects of endothelial progenitor cell related ncRNAs enriched extracellular vesicles on anti-senescence/anti- endothelial dysfunction and recovery of alcoholic liver injury in vivo (Aim 3). At the completion of the proposed research, it is our expectation to have an important positive impact because a mechanistic understanding of the role ncRNAs play in alcohol related injury, endothelial cell and senescence mediated liver disease is likely to provide a foundation for the development of evidence-based clinically useful approaches to treat or prevent human alcoholic liver diseases including US veterans.
Aging is a major risk factor for alcoholic liver diseases and is associated with gradual alteration of liver structure and function as well as significant changes in liver cells including liver sinusoidal endothelial cells. The health relatedness of this grant proposal is that effective treatments are lacking for alcoholic liver diseases that cause vascular dysfunction and senescence in the liver. The rationale for our research is that the successful completion of the studies can ultimately be expected to provide a greater understanding of the functional role of non-coding RNA (ncRNA) mediated endothelial dysfunction and cellular senescence during the progression/recovery of alcoholic liver injuries and increase opportunities for the development of novel treatment paradigms for the management of alcoholic liver diseases.
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