Mechanisms of extrahepatic biliary proliferation and regeneration

项目总结/文摘

PROJECT SUMMARY/ABSTRACT This application seeks an administrative supplement for the ongoing NIDDK K08 funded project. This support will assure completion of the proposed research project with new, complimentary state-of-the-art approaches and facilitate the PI’s transition to the next career stage, an independent NIH-funded investigator. The Supplement will provide needed funds to support progress during the PI’s critical life events, childbirth and primary caregiving responsibilities for her mother. The research project is focused on cholangiopathies, which are incurable, progressive extrahepatic bile duct (EHBD) disorders characterized by injury-induced inflammation and cholangiocyte hyperproliferation. Development of therapies for cholangiopathies requires a better understanding of cellular and molecular mechanisms regulating EHBD proliferation during homeostasis and in response to injury. This supplement and the parent application aim to answer fundamental questions about the mechanisms of EHBD regeneration and explore strategies to modulate biliary proliferation. The rationale for the proposed research is that defining cellular and molecular crosstalk underlying cholangiocyte responses to injury can inform novel therapeutic strategies for cholangiopathies and hepatobiliary regenerative medicine. Our findings to date suggest that Hedgehog (HH) and WNT signaling play important roles in EHBD homeostasis and response to injury. Increased cholangiocyte proliferation after acute injury is associated with an upregulation of cholangiocyte HH ligand expression, which induces stromal cell expression of CXCL1. The CXCL1 cytokine, in turn, recruits neutrophils to the injured tissue to promote biliary proliferation. Additionally, EHBD injury results in WNT signaling upregulation in vivo, and the WNT pathway amplifier, RSPO, is required for EHBD organoid proliferation in vitro. The overarching hypothesis for this and the parent proposal is that HH and WNT signaling regulate crosstalk between epithelial and stromal cells to promote EHBD proliferation after injury. In this proposal I will use wild type and genetic reporter mice to study EHBD cell populations. Bile duct ligation will be used as an injury model. I will examine EHBD cell expression profiles at homeostasis and after injury using single cell and bulk RNA sequencing approaches. These studies will help to (1) determine if GLI1+ HH-responsive cells are fibroblasts expressing Cxcl1; (2) determine if GLI1+ cells are a source of RSPO; and (3) gain insight in obstructive injury-induced expression profiles of epithelial, stromal, and immune cell populations in EHBDs. These experiments are directly pertinent to Aims 1 and 2 of the parent proposal and will increase impact of the K08 project results. Importantly, they will also generate preliminary data for a successful R01 application. Ultimately, the Supplement will allow the PI to step up momentum and promote her transition into an independent NIH-funded investigator and support her long-term goal to understand fundamental mechanisms of EHBD biology to ultimately improve outcomes in patients with cholangiopathies.