HEPATIC WOUND HEALING FOLLOWING ACUTE AND CHRONIC LIVER INJURY: A POTENTIAL ROLE FOR THE HYALURONAN NETWORK
McCracken, Jennifer M.
University of Kansas
Pharmacology, Toxicology & Therapeutics
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Chronic liver disease is the 12th leading cause of death in the United States and consists of a continuum of pathologies. Following initial injury, a patient develops steatosis, or excess fat accumulation. While the liver has the remarkable ability to repair itself, if injury persists, the wound healing process becomes deregulated and excess extracellular matrix (ECM) can accumulate resulting in fibrosis. If the etiologic agent is not removed, a patient can progress to cirrhosis and hepatocellular carcinoma. Despite decades of research, there are currently no effective treatments for advanced liver disease and a liver transplantation is the only treatment option for patients. There are two main approaches of developing novel therapeutics to cure liver disease. First, understanding mechanisms of liver injury can lead to potential pharmacologic targets. Second, because the liver has the remarkable capacity to heal itself, understanding the mechanisms that allow this to happen may reveal potential places that can be targeted to enhance this recovery process. This is an appealing approach because wound healing consists of mostly identical steps regardless of the injury-provoking incident and therefore could apply to liver injury of varied etiologies, and maybe even wound repair in extra-hepatic organs. Therefore, I chose to focus my dissertation research on exploring hepatic wound healing after acute and chronic liver injury. Hyaluronan (HA) is a ubiquitous, anionic glycosaminoglycan in the ECM that can hold 1,000 times its weight in water. HA, HA binding proteins, and HA receptors (the HA network) are implicated in aspects of the wound healing process in the skin, intestine, and lung. The HA network plays roles in injury, inflammation, and fibrogenesis; all primary aspects of wound healing. In fact, high basal HA levels and sustained HA induction in fetal skin are associated with the scarless wound healing that occurs after injury. Adult dermal tissue, which heals with a scar, has lower basal HA levels and a transient HA induction following injury. HA is elevated in the plasma of patients with liver disease and correlates with disease severity. Despite HA’s connection to wound healing and its use as a biomarker for liver disease, little work has been done to investigate if HA is involved in liver wound repair. We hypothesize that the HA network contributes to hepatic wound healing. Our first focus was on a role for HA itself in hepatic wound healing. HA is synthesized by one of three enzymes (HAS1, 2, or 3) and previous studies show that Has3-/- mice have decreased dextran sodium sulfate (DSS)-induced colitis and ventilator induced lung injury. We therefore, hypothesized that Has3-/- mice would be protected from liver injury and fibrosis. Using mice deficient in HAS3, wild-type mice, and carbon tetrachloride (CCl4) we induced both acute and chronic liver injury. We found that Has3-/- mice have increased injury, but also increased wound healing, including inflammation, regeneration, and matrix remodeling compared to wild-type mice after acute CCl4 exposure. This increased wound healing was associated with an increase in hepatic HA deposition and expression of the HA receptor HA mediated motility receptor (HMMR). After chronic CCl4, Has3-/- mice had increased pro-fibrotic transcripts, but no increase in fibrosis. This disconnect was attributed to increased matrix metabolism and was again associated with increased HMMR expression compared to wild-type mice. This led us to investigate a role for HMMR in hepatic wound healing. HMMR can be both intracellular and extracellular and plays a role in cell cycle progression and cell migration, important aspects of wound healing. We hypothesized that Hmmr-/- mice would have delayed wound healing following acute and chronic liver injury due to impaired hepatocyte proliferation and impaired macrophage-mediated matrix metabolism. Using CCl4, we induced both acute and chronic liver injury in Hmmr-/- and wild-type mice. Hmmr-/- mice had decreased inflammation, regeneration, and matrix remodeling despite no difference in liver injury compared to wild-type mice after acute liver injury. This was associated with decreased hepatic HA deposition. After chronic liver injury, Hmmr-/- mice had decreased pro-fibrotic transcripts but no difference in fibrosis. Similar to Has3-/- mice, this disconnect can be attributed to differences in matrix remodeling, with Hmmr-/- mice having less matrix metabolism compared to wild-type mice. My work is the first to demonstrate that HA and HMMR are involved in hepatic wound healing. The culmination of this work led to the development of a working model connecting HA and HMMR with hepatic wound healing. Following liver injury, hepatocytes and resident liver macrophages synthesize pro-inflammatory cytokines and chemokines. These cytokines then stimulate hepatic stellate cells (HSC) to synthesize and deposit HA in the injured areas of the liver, while the chemokines recruit circulating macrophages to the liver. Then, we propose two feed forward loops, the first being where the HA can increase water retention thereby increasing HSC activation by increasing mechanical stress and further increasing HA deposition. Second, we also propose a feed forward loop between HA and inflammation, where the HA contributes to inflammation and increased inflammation further increases HA synthesis by HSC. Additionally, we propose that macrophages that are being recruited to the liver express HMMR and can use the HA:HMMR interaction to localize to the necrotic area and clear tissue debris. In the case of chronic liver injury, this HA:HMMR interaction can localize scar associated macrophages to the fibrotic septae and increase matrix degradation and facilitate fibrosis resolution. Finally, the increased inflammatory microenvironment of the healing liver, and perhaps intracellular HMMR, promotes hepatocyte proliferation. Together, HA and HMMR contributes to inflammation, regeneration, and matrix remodeling to aid in the hepatic wound healing process. Taken together, our data demonstrate that the HA network, in particular HA and HMMR, is involved in both acute and chronic liver injury and wound healing. Further work should be completed to evaluate other aspects of the HA network, including other receptors and HA binding proteins. Additionally, exploring what role the HA:HMMR interaction plays in acute and chronic liver injury should be explored further.
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