The role of extracellular matrix in primary sclerosing cholangitis

Veterans Admin Palo Alto Health Care Sys, Palo Alto CA

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Abstract

Primary sclerosing cholangitis (PSC), is a devastating cholestatic liver disease, characterized by segmental inflammation and fibrosis of the bile ducts. PSC still has no accepted therapies, and the VA population is increasingly affected. The extracellular matrix (ECM), and hyaluronan (HA), is abundant in PSC, surrounding the affected areas. The mechanism by which HA modulates the ECM in early PSC, mechano-cellular and immune responses, are unknown. In our studies, we found that HA is abundant in patients with PSC and in mouse models prior to the deposition of collagen, and that high molecular weight (HMW) HA induces matrix stiffening, tested in a novel 3D adjustable stiffness hydrogel model, and in DDC diet by atomic force microscopy (AFM). We depicted CD44/Integrin-β1 as mechanoreceptors that transmit YAP-mediated proliferative responses and a ductular reactive cell (DRC) phenotype. Furthermore, treatment of DDC-fed or Mdr2-/- mice with 4- methylumbelliferone gluconate (4-MUG), an inhibitor of HA synthesis, reduced stiffness, and downregulated pro- inflammatory cytokines. Thus, we hypothesize that the changing ECM architecture and mechano-signaling elicit phenotypic changes in cholangiocytes as well as modulate macrophage polarity and effector functions in PSC. We propose three SPECIFIC AIMS to address the key areas generated by this hypothesis: Our first aim is to determine the role of hyaluronan modulating early matrix changes and mechano-signaling in PSC. 1) We propose to study the role of high and low molecular weight-HA in mouse models of PSC by second harmonic generation and CT-FIRE microcopy, as well as matrix mechanics (rheometry, AFM), in 3D alginate/collagen hydrogels. 2) We will investigate mechanosensitive pathways, focusing on CD44 and integrin β1 interaction and the role of YAP/TAZ activation in cytoskeletal rearrangement, proliferative responses and hyaluronan synthetase (HAS1, 2) production in an autocrine cycle. 3) ECM changes, mechano-signaling, and DRC will be studied in models of PSC, combined with HAS inhibition. Cell-specific mechano-signals will be evaluated in a novel conditional, inducible cholangiocyte CD44KO model (CD44 fl/fl X Opn-iCreERT2 mice), on DDC diet. In aim 2 we will explore how the stiffening matrix in PSC affects macrophage phenotypic and functional behavior. 1) We will evaluate the mechanism of podosome formation, mechano-sensation and migratory activity linked to phenotypic changes. 2)The effects of stiffening matrix on macrophage phagocytosis and efferocytosis will be assessed. The molecular events of phagosome maturation and expression of “don’t eat me signals” will be evaluated. 3) Monocyte/macrophage mobilization, trafficking, and fate will be studied in the hCD68-GFP transgenic mice on DDC diet. The dynamics of GFP-labeled cells will be studied by CyTOF during disease progression. In the third aim, the effects of HAS inhibition will be tested in PSC models, and in patient liver samples. 1) We will study the dynamic changes elicited by HAS inhibition on inflammation and fibrosis. We will garner information at a single cell level, using Codex, assessing the dynamics of cell interaction across the entire landscape. 2) De-identified biopsy samples from the Stanford Tissue Bank and a pilot PSC trial using 4-MU vs. standard of care will be obtained. AFM will be performed on the peri-biliary areas. To explore cholangiocyte and macrophage plasticity/interactome, we will use a spatial transcriptomic platform (Visium). These studies will fill the gap in knowledge regarding matrix-mediated regulation of cell plasticity and elucidate early changes in PSC. HAS targeting could become a feasible an effective approach to halt disease progression.