A fetal wound healing program after intrauterine bile duct injury may contribute to biliary atresia

Abstract

Background & aims: Biliary atresia (BA) is an obstructive cholangiopathy that initially affects the extrahepatic bile ducts (EHBDs) of neonates. The etiology is uncertain, but evidence points to a prenatal cause. Fetal tissues have increased levels of hyaluronic acid (HA), which plays an integral role in fetal wound healing. The objective of this study was to determine whether a program of fetal wound healing is part of the response to fetal EHBD injury.

Methods: Mouse, rat, sheep, and human EHBD samples were studied at different developmental time points. Models included a fetal sheep model of prenatal hypoxia, human BA EHBD remnants and liver samples taken at the time of the Kasai procedure, EHBDs isolated from neonatal rats and mice, and spheroids and other models generated from primary neonatal mouse cholangiocytes.

Results: A wide layer of high molecular weight HA encircling the lumen was characteristic of the normal perinatal but not adult EHBD. This layer, which was surrounded by collagen, expanded in injured ducts in parallel with extensive peribiliary gland hyperplasia, increased mucus production and elevated serum bilirubin levels. BA EHBD remnants similarly showed increased HA centered around ductular structures compared with age-appropriate controls. High molecular weight HA typical of the fetal/neonatal ducts caused increased cholangiocyte spheroid growth, whereas low molecular weight HA induced abnormal epithelial morphology; low molecular weight HA caused matrix swelling in a bile duct-on-a-chip device.

Conclusion: The fetal/neonatal EHBD, including in human EHBD remnants from Kasai surgeries, demonstrated an injury response with prolonged high levels of HA typical of fetal wound healing. The expanded peri-luminal HA layer may swell and lead to elevated bilirubin levels and obstruction of the EHBD.

Impact and implications: Biliary atresia is a pediatric cholangiopathy associated with high morbidity and mortality rates; although multiple etiologies have been proposed, the fetal response to bile duct damage is largely unknown. This study explores the fetal pathogenesis after extrahepatic bile duct damage, thereby opening a completely new avenue to study therapeutic targets in the context of biliary atresia.

Keywords: biliary atresia; cholangiocyte; cholangiopathy; hyaluronic acid; peribiliary glands.

Fig. 1.. Normal mammalian fetal and neonatal EHBDs have high HA and low collagen compared to the adult.

(A) HA content of adult or fetal/neonatal human (GA: 22–39 weeks), sheep (GA: 121 and 128 days), and rat and mouse EHBDs (DOL 1). HA content was defined as % coverage of wall by HA binding protein signal (n ≥ 3 individuals). (B) Concentration of HA (ng) per mg wet weight EHBD. n = 3 homogenates, each containing 2–15 EHBDs. (C) Ratio of HA and collagen in the EHBD, normalized to DOL15. (D) Human fetus (GA: 36 weeks) and adult EHBD. SHG imaging includes both forward and backward scatter (E) Sheep fetus (GA: 121 days) and adult EHBD. All graphs show mean±SD. Significance determined by Student’s t test (A) or One-Way ANOVA with Tukey’s post-hoc test (B).

Fig. 2.. Prenatal EHBD injury causes an increase in HA.

(A) HA layer of damaged/diseased and normal fetal sheep (GA: 128 days) and infant and adult human EHBDs. Significance determined by Student’s t-test (fetal sheep) and Mann-Whitney test (infant and adult human). (B) Representative images of n = 9 EHBD BA remnants. Overlay of immunohistochemistry for HA (green) and visualization of collagen by SHG imaging (forward and backward scatter are both magenta) and Sirius Red staining. Graphs show mean±SD of n ≥ 3 individuals for each condition. All scale bars = 50 μm.

Fig. 3.. Collagen in BA is organized around HA areas and the fibers are shorter.

(A) Representative images of n = 14 BA remnants, with collagen visualized by SHG imaging and HA by HA binding protein. (B) Mask rendering from SHG signals (both forward and backward scatter) on a control sample (age: 2 months) (C) Schematic of curvature and endpoint calculations. (D) Representative SHG imaging of EHBDs from human controls (n = 5; age: 2–6 months) and BA remnants (n = 14) taken at the Kasai procedure. (E) Fiber curvature, endpoints, length, and density. Significance determined by Student’s t test. All scale bars = 50 μm.

Fig. 4.. Prenatal hypoxia causes a marked epithelial regenerative response in EHBDs of fetal sheep.

(A) H&E staining of EHBDs from control (n = 6) and hypoxia (n = 8) groups (GA: 121 and 128 days) and graphs representing PBG area. (B) Immunofluorescence for Ki-67 (proliferation), K19 (cholangiocytes), and vimentin (mesenchymal cells) on sheep fetus EHBDs (GA 121 and 128). n = 4 (controls) and n = 8 (hypoxia). (C) Representative HA stains (brown) in control and hypoxia groups (n ≥ 3 for each timepoint). (D) HA layer width (from C) and PBG area (from A). Graphs show mean±SE of the best curve to fit the data. (E) Correlation between the HA layer width and PBG area (as plotted in D). (F) Human control EHBDs (n = 5; age: 2–6 months) and BA remnants (n = 14) stained for PDX1, AE2, Sox9, and Ki-67. %positive cells is provided. Significance determined by Student’s t-test (A-D), Mann-Whitney (B:cholangiocytes) or Spearman correlation (E). Graph shows mean±SD (all panels). All scale bars = 50 μm.

Fig. 5.. HA deposition predisposes to obstruction.

(A) H&E staining of a representative fetal sheep liver in the hypoxia group showing bile plugs. Serum bilirubin levels of fetuses in the control (n = 10) and hypoxia (n = 6) groups (GA: 121 and 128 days). (B) SHG imaging of EHBDs from control (n = 7) and hypoxic fetal sheep (n = 8) to determine collagen density and width of collagen layers. (C) Setup of the microfluidic device. (D). (left) Devices with the three types of matrices (n = 3, each), before and after swelling. (right) Violin plot of % change in lumen diameter for each group. Significance determined by Student’s t-test or One-Way ANOVA with Tukey’s post-hoc test (D). Graphs show mean±SD. All scale bars = 50 μm.

Fig. 6.. The fetal EHBD regenerative response involves an increase in mucus production.

(A) PAS and alcian blue-stained EHBDs from fetal sheep in control and hypoxia groups. Table indicates % PBGs showing mucus production for the indicated gestational age (n ≥ 3 for each timepoint). Colors, from light to dark purple, correspond to increasing percentages (<15%, <20%, and >20%). (B) Combined PAS and alcian blue staining of goblet cells in the surface epithelium of the hypoxia group. (C) PAS staining of bile in the gallbladder (controls did not show intrahepatic bile plugs) and bile plug in the intrahepatic bile ducts (D) PAS staining of intrahepatic bile plugs of patients with PSC or BA (n = 3, each).

Fig. 7.. HA size distribution and production/degradation enzymes differ between neonates, adults, and after injury.

(A) Mouse neonatal EHBDs at day 2 after birth and adult EHBDs stained for HAS1–3 and HYAL1–2. Percentage positive mesenchymal cells for n=3 for each stain shown in graph. (B) HA size distribution in mouse and rat neonatal and adult EHBDs, as measured by solid-state nanopore technology. (C) HA in situ zymography using fluorescein-labelled HA, overlaid on 4/5 day-old rat neonatal EHBDs after immediate harvest (control; n = 5) or after an additional 5-day treatment with DMSO (n = 6) or biliatresone (n = 6). Significance determined by Student’s t-test or One-Way ANOVA with Tukey’s post-hoc test (C). Graphs show mean ± SD (all panels).

Fig. 8.. LMW and HMW have different effects on EHBD cholangiocytes and mesenchymal cells.

(A) Spheroids derived from mouse primary cholangiocytes cultured in collagen with PBS, LMW HA, or HMW HA (n = 5 biological repeats with 2–3 technical replicates per condition). (B) Proliferation (Ki-67) of spheroids in each condition. (C) Spheroids stained with F-actin with bright field background. (D) Morphological scoring of spheroids in each condition. (E) Human cholangiocytes were seeded in the bile duct-on-a-chip device with a mixture of collagen alone or HMW HA with collagen in the ECM compartment. Immunofluorescence for Ki-67 on the right (n = 3 biological repeats with 2 technical replicates per condition). (F) (left) K19, αSMA, and vimentin staining of 2D co-cultures. (middle) SHG imaging (back scatter) of the cholangiocyte/fibroblast-derived matrices in each condition (n = 3 biological repeats with 3 technical replicates per condition). Significance determined by area under the curve (A), Student’s t-test (B, E), Chi square test (D) and One-way and Two-way ANOVA for SHG intensity and collagen alignment, respectively (F). Graphs show mean ± SD (all panels). All scale bars = 50 μm.