Biliary atresia: insights into mechanisms using a toxic model of the disease including Wnt and Hippo signaling pathways and microtubules.

BACKGROUND: Mechanisms underlying bile duct injury in biliary atresia (BA) remain unclear and mechanisms of bile duct repair are unknown. This study aimed to explore the roles of microtubule instability and Wnt and Hippo signaling pathways in a biliatresone-induced BA model. METHODS: Using primary murine neonatal cholangiocytes in both 2D and 3D cultures, and ex-vivo extra hepatic bile ducts (EHBD) which also has peri-cholangiocyte area, we analyzed injury and recovery processes. Injury was induced by the toxin biliatresone and recovery was induced by toxin wash-out. RESULTS: Microtubule stabilizer paclitaxel prevented biliatresone-induced injury, both to cholangiocytes as well as reduced periductal αSMA stain, this process is mediated by decreased glutathione levels. RhoU and Wnt11 (Wnt signaling) and Pard6g and Amotl1 (Hippo signaling) are involved in both injury and recovery processes, with the latter acting upstream to Wnt signaling. CONCLUSIONS: Early stages of biliatresone-induced EHBD injury in cholangiocytes and periductal structures are reversible. Wnt and Hippo signaling pathways play crucial roles in injury and recovery, providing insights into BA injury mechanisms and potential recovery avenues. IMPACT: Microtubule stabilization prevents cholangiocyte injury and lumen obstruction in a toxic model of biliary atresia (biliatresone induced). Early stages of biliatresone-induced injury, affecting both cholangiocytes and periductal structures, are reversible. Both Wnt and Hippo signaling pathways play a crucial role in bile duct injury and recovery, with a noted interplay between the two. Understanding mechanisms of cholangiocyte recovery is imperative to unveil potential therapeutic avenues.

In Utero Extrahepatic Bile Duct Damage and Repair: Implications for Biliary Atresia.

Biliary atresia (BA) is a cholangiopathy affecting the extrahepatic bile duct (EHBD) of newborns. The etiology and pathophysiology of BA are not fully understood; however, multiple causes of damage and obstruction of the neonatal EHBD have been identified. Initial damage to the EHBD likely occurs before birth. We discuss how different developmental stages in utero and birth itself could influence the susceptibility of the fetal EHBD to damage and a damaging wound-healing response. We propose that a damage-repair response of the fetal and neonatal EHBD involving redox stress and a program of fetal wound healing could-regardless of the cause of the initial damage-lead to either obstruction and BA or repair of the duct and recovery. This overarching concept should guide future research targeted toward identification of factors that contribute to recovery as opposed to progression of injury and fibrosis. Viewing BA through the lens of an in utero damage-repair response could open up new avenues for research and suggests exciting new therapeutic targets.

On the relationship between viscoelasticity and water diffusion in soft biological tissues.

Magnetic resonance elastography (MRE) and diffusion-weighted imaging (DWI) are complementary imaging techniques that detect disease based on viscoelasticity and water mobility, respectively. However, the relationship between viscoelasticity and water diffusion is still poorly understood, hindering the clinical translation of combined DWI-MRE markers. We used DWI-MRE to study 129 biomaterial samples including native and cross-linked collagen, glycosaminoglycans (GAGs) with different sulfation levels, and decellularized specimens of pancreas and liver, all with different proportions of solid tissue, or solid fractions. We developed a theoretical framework of the relationship between mechanical loss and tissue-water mobility based on two parameters, solid and fluid viscosity. These parameters revealed distinct DWI-MRE property clusters characterizing weak, moderate, and strong water-network interactions. Sparse networks interacting weakly with water, such as collagen or diluted decellularized tissue, resulted in marginal changes in water diffusion over increasing solid viscosity. In contrast, dense networks with larger solid fractions exhibited both free and hindered water diffusion depending on the polarity of the solid components. For example, polar and highly sulfated GAGs as well as native soft tissues hindered water diffusion despite relatively low solid viscosity. Our results suggest that two fundamental properties of tissue networks, solid fraction and network polarity, critically influence solid and fluid viscosity in biological tissues. Since clinical DWI and MRE are sensitive to these viscosity parameters, the framework we present here can be used to detect tissue remodeling and architectural changes in the setting of diagnostic imaging. STATEMENT OF SIGNIFICANCE: The viscoelastic properties of biological tissues provide a wealth of information on the vital state of cells and host matrix. Combined measurement of viscoelasticity and water diffusion by medical imaging is sensitive to tissue microarchitecture. However, the relationship between viscoelasticity and water diffusion is still poorly understood, hindering full exploitation of these properties as a combined clinical biomarker. Therefore, we analyzed the parameter space accessible by diffusion-weighted imaging (DWI) and magnetic resonance elastography (MRE) and developed a theoretical framework for the relationship between water mobility and mechanical parameters in biomaterials. Our theory of solid material properties related to particle motion can be translated to clinical radiology using clinically established MRE and DWI.

Low-dose biliatresone treatment of pregnant mice causes subclinical biliary disease in their offspring: Evidence for a spectrum of neonatal injury.

Biliary atresia is a neonatal disease characterized by damage, inflammation, and fibrosis of the liver and bile ducts and by abnormal bile metabolism. It likely results from a prenatal environmental exposure that spares the mother and affects the fetus. Our aim was to develop a model of fetal injury by exposing pregnant mice to low-dose biliatresone, a plant toxin implicated in biliary atresia in livestock, and then to determine whether there was a hepatobiliary phenotype in their pups. Pregnant mice were treated orally with 15 mg/kg/d biliatresone for 2 days. Histology of the liver and bile ducts, serum bile acids, and liver immune cells of pups from treated mothers were analyzed at P5 and P21. Pups had no evidence of histological liver or bile duct injury or fibrosis at either timepoint. In addition, growth was normal. However, serum levels of glycocholic acid were elevated at P5, suggesting altered bile metabolism, and the serum bile acid profile became increasingly abnormal through P21, with enhanced glycine conjugation of bile acids. There was also immune cell activation observed in the liver at P21. These results suggest that prenatal exposure to low doses of an environmental toxin can cause subclinical disease including liver inflammation and aberrant bile metabolism even in the absence of histological changes. This finding suggests a wide potential spectrum of disease after fetal biliary injury.

Metabolically intact nuclei are fluidized by the activity of the chromatin remodeling motor BRG1.

The structure and dynamics of the cell nucleus regulate nearly every facet of the cell. Changes in nuclear shape limit cell motility and gene expression. Although the nucleus is generally seen as the stiffest organelle in the cell, cells can nevertheless deform the nucleus to large strains by small mechanical stresses. Here, we show that the mechanical response of the cell nucleus exhibits active fluidization that is driven by the BRG 1 motor of the SWI/SNF/BAF chromatin-remodeling complex. Atomic force microscopy measurements show that the nucleus alters stiffness in response to the cell substrate stiffness, which is retained after the nucleus is isolated and that the work of nuclear compression is mostly dissipated rather than elastically stored. Inhibiting BRG 1 stiffens the nucleus and eliminates dissipation and nuclear remodeling both in isolated nuclei and in intact cells. These findings demonstrate a novel link between nuclear motor activity and global nuclear mechanics.

The ß-Secretase BACE1 Drives Fibroblast Activation in Systemic Sclerosis through the APP/ß-Catenin/Notch Signaling Axis.

BACE1 is well-known for its role in the development of Alzheimer's disease. Recent publications, including our own, have demonstrated a role for this enzyme in other chronic diseases. The aim of this study was to investigate the role of BACE1 in the autoimmune disease systemic sclerosis (SSc). BACE1 protein levels were elevated in the skin of patients with SSc. Inhibition of BACE1 with small-molecule inhibitors or small interfering RNA blocked SSc and fibrotic stimuli-mediated fibroblast activation. Furthermore, we show that BACE1 regulation of dermal fibroblast activation is dependent on ß-catenin and Notch signaling. The neurotropic factor brain-derived neurotrophic factor negatively regulates BACE1 expression and activity in dermal fibroblasts. Finally, sera from patients with SSc show higher ß-amyloid and lower brain-derived neurotrophic factor levels than healthy controls. The ability of BACE1 to regulate SSc fibroblast activation reveals a therapeutic target in SSc. Several BACE1 inhibitors have been shown to be safe in clinical trials for Alzheimer's disease and could be repurposed to ameliorate fibrosis progression.

Toxins and Biliary Atresia: Is Karenia Brevis (Red Tide) The Culprit?

OBJECTIVE: The study objective was to evaluate the association between Karenia brevis (K. brevis) exposure during pregnancy and the prevalence of biliary atresia (BA) in offspring. STUDY DESIGN: This was a hospital-based, case-control study in which cases were infants diagnosed with BA at Johns Hopkins All Children's Hospital from October 2001 to December 2019. Cases were matched 1:4 by age to controls who were randomly selected from a pool of healthy infants hospitalized during the study period for common pediatric diagnoses. Infants were excluded if they had congenital anomalies and/or were non-Florida residents. Gestational K. brevis exposure levels (cells/liter) were determined from Florida Fish and Wildlife Conservation Commission exposure data at 10- and 50 mile radii from the mother's zip code of residence. Multivariable conditional logistic regression determined odds of BA in offspring in relation to maternal gestational K. brevis exposure adjusted for infant sex, race/ethnicity, coastal residence, and seasonality. RESULTS: Of 38 cases and 152 controls, no significant inter-group differences were observed for infant race/ethnicity, season of birth, or coastal residence. Median gestational exposure at the 10 mile radius was 0 cells/liter in both groups. A greater proportion of cases had no gestational K. brevis exposure (63.2 %, n = 24) in comparison to controls (37.5 %, n = 57; p = .04) at a 10 mile radius. At a 50 mile radius, cases had a peak median exposure at 6 months of gestation compared to controls' peak at 9 months. After adjustment for sex, seasonality, race/ethnicity, and coastal residence, there was no significant association between BA and maximum K. brevis exposure per trimester of pregnancy observed at a 10- or 50 mile radius. CONCLUSION: In this matched case-control study, we observed no association between gestational K. brevis (cells/liter) exposure at a 10- or 50 mile radius from maternal zip code of residence and BA in offspring.

Matrix viscoelasticity promotes liver cancer progression in the pre-cirrhotic liver.

Type 2 diabetes mellitus is a major risk factor for hepatocellular carcinoma (HCC). Changes in extracellular matrix (ECM) mechanics contribute to cancer development1,2, and increased stiffness is known to promote HCC progression in cirrhotic conditions3,4. Type 2 diabetes mellitus is characterized by an accumulation of advanced glycation end-products (AGEs) in the ECM; however, how this affects HCC in non-cirrhotic conditions is unclear. Here we find that, in patients and animal models, AGEs promote changes in collagen architecture and enhance ECM viscoelasticity, with greater viscous dissipation and faster stress relaxation, but not changes in stiffness. High AGEs and viscoelasticity combined with oncogenic ß-catenin signalling promote HCC induction, whereas inhibiting AGE production, reconstituting the AGE clearance receptor AGER1 or breaking AGE-mediated collagen cross-links reduces viscoelasticity and HCC growth. Matrix analysis and computational modelling demonstrate that lower interconnectivity of AGE-bundled collagen matrix, marked by shorter fibre length and greater heterogeneity, enhances viscoelasticity. Mechanistically, animal studies and 3D cell cultures show that enhanced viscoelasticity promotes HCC cell proliferation and invasion through an integrin-ß1-tensin-1-YAP mechanotransductive pathway. These results reveal that AGE-mediated structural changes enhance ECM viscoelasticity, and that viscoelasticity can promote cancer progression in vivo, independent of stiffness.

Usual Source of Care and Contraceptive Use.

BACKGROUND: A high proportion of people in the United States at risk of unintended pregnancy also have limited primary care access. STUDY DESIGN: We pooled data for analyses from separate 2015-2017 and 2017-2019 waves of the National Survey of Family Growth. Multivariable logistic regression was used to estimate associations between the usual source of health care and self-reported use of a comprehensive range of contraceptive options, as well as alignment between patient preference and the current method. RESULTS: Compared with having a private doctor or Health Maintenance Organization, not having a usual source of care was associated with lower odds of using short-term hormonal methods (OR=0.54, 95% CI: 0.40-0.73, for an 11 percentage point lower rate); higher odds of using time-based methods (OR=1.47, 95% CI: 1.10-1.97, for a 6 percentage point higher rate); and higher odds of preferring a contraceptive method other than the one most recently used (OR=1.39, 95% CI: 1.01-1.90, for a 6 percentage point higher probability). Reliance on an emergency department as a usual source of care was not associated with contraceptive use or satisfaction with the method used. Reliance on urgent care was associated only with higher odds of using time-based methods (OR=1.60, 95% CI: 1.03-2.50, for a 7 percentage point higher rate). Clinic-based usual care was not associated with any differences in contraceptive use but was associated with preferring a contraceptive method other than the one most recently used (OR=1.65, 95% CI: 1.21-2.25, for an 8 percentage point higher probability). CONCLUSIONS: All sources of usual care can improve contraceptive access.

Contraception Use by Title X Clients and Clients of Other Providers, 2015-2019.

INTRODUCTION: Title X clinics provide access to a wide range of contraceptive options for individuals of all income levels and documentation statuses. As Title X continues to face political uncertainties, it is important to provide up-to-date information about its clients' use of contraception. This study used recent nationally representative data to compare contraception received by Title X clients with that received by clients of other providers. METHODS: This article draws on 2015-2017 and 2017-2019 waves of the National Survey of Family Growth. The sample was restricted to 15- to 44-year-old women needing contraception. Logistic regressions estimated associations between receiving services at Title X clinics versus at other providers (including private) and use of a range of contraceptive options, as well as number of months' supply for those using oral contraceptives. RESULTS: In 2015-2017, Title X was associated with using any contraception (adjusted odds ratio [AOR], 4.11; p = .004). In both waves, Title X clients were more likely to use long-acting reversible contraceptives (AOR, 1.78 in 2015-2017 [p = .023] and AOR, 2.59 in 2017-2019 [p = .003]) and hormonal methods other than oral contraceptives (AOR, 2.31 in 2015-2017 [p = .007] and AOR, 3.04 in 2017-2019 [p = .001]). In both waves, Title X clients using oral contraceptives were also more likely than non-Title X clients to receive more than a 3-month supply (AOR, 3.54 in 2015-2017 [p = .008] and AOR, 2.61 in 2017-2019 [p = .043]). Title X was not associated in either wave with use of barrier or time-based methods, such as periodic abstinence or withdrawal. CONCLUSIONS: Patterns of contraceptive use by Title X clients compared with those of clients of other providers indicate that the Title X program has allowed access to a wide range of contraceptive methods. Ongoing research is necessary to see whether these patterns change over time.

Conjugated hyperbilirubinemia is associated with increased morbidity and mortality after neonatal heart surgery.

BACKGROUND: Cholestasis characterised by conjugated hyperbilirubinemia is a marker of hepatobiliary dysfunction following neonatal cardiac surgery. We aimed to characterise the incidence of conjugated hyperbilirubinemia following neonatal heart surgery and examine the effect of conjugated hyperbilirubinemia on post-operative morbidity and mortality. METHODS: This was a retrospective study of all neonates who underwent surgery for congenital heart disease (CHD) at our institution between 1/1/2010 and 12/31/2020. Patient- and surgery-specific data were abstracted from local registry data and review of the medical record. Conjugated hyperbilirubinemia was defined as perioperative maximum conjugated bilirubin level > 1 mg/dL. The primary outcome was in-hospital mortality. Survival analysis was conducted using the Kaplan-Meier survival function. RESULTS: Conjugated hyperbilirubinemia occurred in 8.5% of patients during the study period. Neonates with conjugated hyperbilirubinemia were more likely to be of younger gestational age, lower birth weight, and non-Caucasian race (all p < 0.001). Patients with conjugated hyperbilirubinemia were more likely to have chromosomal and non-cardiac anomalies and require ECMO pre-operatively. In-hospital mortality among patients with conjugated hyperbilirubinemia was increased compared to those without (odds ratio 5.4). Post-operative complications including mechanical circulatory support, reoperation, prolonged ventilator dependence, and multi-system organ failure were more common with conjugated hyperbilirubinemia (all p < 0.04). Patients with higher levels of conjugated bilirubin had worst intermediate-term survival, with patients in the highest conjugated bilirubin group (>10 mg/dL) having a 1-year survival of only 6%. CONCLUSIONS: Conjugated hyperbilirubinemia is associated with post-operative complications and worse survival following neonatal heart surgery. Cholestasis is more common in patients with chromosomal abnormalities and non-cardiac anomalies, but the underlying mechanisms have not been delineated.

Understanding the use of media analysis in public health research through food tax debates (HEALTHEI Project): a scoping review.

BACKGROUND: Poor diet is a major public health concern. In 2021, 63·8% of adults and 22·2% of reception-age children were either overweight or obese in England. Fiscal interventions have become a popular policy measure to reduce obesity and encourage healthy eating. Such measures are highly controversial, leading to media debate promoting pro-tax and anti-tax arguments. To better understand food tax debates and the use of media analysis in public health research, we conducted a scoping review of media analyses using food taxes as a case study. METHODS: In this scoping review, we searched SCOPUS, PubMed, and EBSCOhost databases on Feb 14-22, 2023, using keyword variations for "food", "tax", and "media analysis". Results were restricted to English-only, peer-reviewed journal articles. The initial results were manually screened through an iterative process to exclude articles that did not analyse a food tax, were non-English language, were not peer-reviewed, or did not use media analysis as the primary method. Modelled on Arksey and O'Malley's (2005) five-stage review protocol, two researchers used a coding framework to independently code all articles and checked result quality through regular discussion. Extracted data included article title, author, year, country, tax type, media sources used, identified media frames, and research aims, methods, results, and conclusions. Results are reported according to PRISMA guidelines and data files submitted to FigShare Repository (non-accessible). FINDINGS: Of 1087 articles reviewed, 19 were eligible to be included in the study. Articles were published between 2013 and 2023, with 2021 having the highest concentration of studies carried out mainly in UK and USA. Despite search terms encompassing a range of food products, the retrieved media analyses focused on three types of food product taxes: sugar-sweetened beverages, meat, and groceries. Most articles explored arguments for and against policy implementation, with some investigating stakeholder representation. Results demonstrate that stakeholders' arguments, both positive and negative, are consistent across countries and food products. INTERPRETATION: The consistency of how both pro-tax and anti-tax arguments are presented in the media demonstrates the importance of coordination between stakeholder groups to influence policy adoption. To our knowledge, this is the first study to investigate media analysis across a diverse range of food products. FUNDING: National Institute for Health and Care Research (NIHR).

Human vascularized bile duct-on-a chip: a multi-cellular micro-physiological system for studying cholestatic liver disease.

Exploring the pathogenesis of and developing therapies for cholestatic liver diseases such as primary sclerosing cholangitis (PSC) remains challenging, partly due to a paucity ofin vitromodels that capture the complex environments contributing to disease progression and partly due to difficulty in obtaining cholangiocytes. Here we report the development of a human vascularized bile duct-on-a-chip (VBDOC) that uses cholangiocyte organoids derived from normal bile duct tissue and human vascular endothelial cells to model bile ducts and blood vessels structurally and functionally in three dimensions. Cholangiocytes in the duct polarized, formed mature tight junctions and had permeability properties comparable to those measured inex vivosystems. The flow of blood and bile was modeled by perfusion of the cell-lined channels, and cholangiocytes and endothelial cells displayed differential responses to flow. We also showed that the device can be constructed with biliary organoids from cells isolated from both bile duct tissue and the bile of PSC patients. Cholangiocytes in the duct became more inflammatory under the stimulation of IL-17A, which induced peripheral blood mononuclear cells and differentiated Th17 cells to transmigrate across the vascular channel. In sum, this human VBDOC recapitulated the vascular-biliary interface structurally and functionally and represents a novel multicellular platform to study inflammatory and fibrotic cholestatic liver diseases.

Microcystin-RR is a biliary toxin selective for neonatal cholangiocytes.

BACKGROUND AND AIMS: Biliary atresia is a fibrosing cholangiopathy affecting neonates that is thought to be caused by a prenatal environmental insult to the bile duct. Biliatresone, a plant toxin with an α-methylene ketone group, was previously implicated in toxin-induced biliary atresia in Australian livestock, but is found in a limited location and is highly unlikely to be a significant human toxin. We hypothesized that other molecules with α-methylene ketone groups, some with the potential for significant human exposure, might also be biliary toxins. APPROACH AND RESULTS: We focused on the family of microcystins, cyclic peptide toxins from blue-green algae that have an α-methylene ketone group and are found worldwide, particularly during harmful algal blooms. We found that microcystin-RR, but not 6 other microcystins, caused damage to cell spheroids made using cholangiocytes isolated from 2-3-day-old mice, but not from adult mice. We also found that microcystin-RR caused occlusion of extrahepatic bile duct explants from 2-day-old mice, but not 18-day-old mice. Microcystin-RR caused elevated reactive oxygen species in neonatal cholangiocytes, and treatment with N-acetyl cysteine partially prevented microcystin-RRinduced lumen closure, suggesting a role for redox homeostasis in its mechanism of action. CONCLUSIONS: This study highlights the potential for environmental toxins to cause neonatal biliary disease and identifies microcystin-RR acting via increased redox stress as a possible neonatal bile duct toxin.

Differences in Use of Clinical Decision Support Tools and Implementation of Aspirin, Blood Pressure Control, Cholesterol Management, and Smoking Cessation Quality Metrics in Small Practices by Race and Sex.

Importance: Practice-level evidence is needed to clarify the value of population-based clinical decision support (CDS) tools in reducing racial and sex disparities in cardiovascular care. Objective: To evaluate the association between CDS tools and racial and sex disparities in the aspirin use, blood pressure control, cholesterol management, and smoking cessation (ABCS) care quality metrics among smaller primary care practices. Design, Setting, and Participants: This cross-sectional study used practice-level data from the Agency for Healthcare Research and Quality-funded EvidenceNOW initiative. The national initiative from May 1, 2015, to April 30, 2021, spanned 12 US states and focused on improving cardiovascular preventive care by providing quality improvement support to smaller primary care practices. A total of 576 primary care practices in EvidenceNOW submitted both survey data and electronic health record (EHR)-derived ABCS data stratified by race and sex. Main Outcomes and Measures: Practice-level estimates of disparities between Black and White patients and between male and female patients were calculated as the difference in proportions of eligible patients within each practice meeting ABCS care quality metrics. The association between CDS tools (EHR prompts, standing orders, and clinical registries) and disparities was evaluated by multiply imputed multivariable models for each CDS tool, adjusted for practice rurality, ownership, and size. Results: Across the 576 practices included in the analysis, 219 (38.0%) had patient panels that were more than half White and 327 (56.8%) had panels that were more than half women. The proportion of White compared with Black patients meeting metrics for blood pressure (difference, 5.16% [95% CI, 4.29%-6.02%]; P < .001) and cholesterol management (difference, 1.49% [95% CI, 0.04%-2.93%] P = .04) was higher; the proportion of men meeting metrics for aspirin use (difference, 4.36% [95% CI, 3.34%-5.38%]; P < .001) and cholesterol management (difference, 3.88% [95% CI, 3.14%-4.63%]; P < .001) was higher compared with women. Conversely, the proportion of women meeting practice blood pressure control (difference, -1.80% [95% CI, -2.32% to -1.28%]; P < .001) and smoking cessation counseling (difference, -1.67% [95% CI, -2.38% to -0.95%]; P < .001) metrics was higher compared with men. Use of CDS tools was not associated with differences in race or sex disparities except for the smoking metric. Practices using CDS tools showed a higher proportion of men meeting the smoking counseling metric than women (coefficient, 3.82 [95% CI, 0.95-6.68]; P = .009). Conclusions and Relevance: The findings of this cross-sectional study suggest that practices using CDS tools had small disparities that were not statistically significant, but CDS tools were not associated with reductions in disparities. More research is needed on effective practice-level interventions to mitigate disparities.

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

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.