A Bile Duct-on-a-Chip With Organ-Level Functions.

BACKGROUND AND AIMS: Chronic cholestatic liver diseases, such as primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC), are frequently associated with damage to the barrier function of the biliary epithelium. Here, we report on a bile duct-on-a-chip that phenocopies not only the tubular architecture of the bile duct in three dimensions, but also its barrier functions. APPROACH AND RESULTS: We showed that mouse cholangiocytes in the channel of the device became polarized and formed mature tight junctions, that the permeability of the cholangiocyte monolayer was comparable to ex vivo measurements, and that cholangiocytes in the device were mechanosensitive (as demonstrated by changes in calcium flux under applied luminal flow). Permeability decreased significantly when cells formed a compact monolayer with cell densities comparable to those observed in vivo. This device enabled independent access to the apical and basolateral surfaces of the cholangiocyte channel, allowing proof-of-concept toxicity studies with the biliary toxin, biliatresone, and the bile acid, glycochenodeoxycholic acid. The cholangiocyte basolateral side was more vulnerable than the apical side to treatment with either agent, suggesting a protective adaptation of the apical surface that is normally exposed to bile. Further studies revealed a protective role of the cholangiocyte apical glycocalyx, wherein disruption of the glycocalyx with neuraminidase increased the permeability of the cholangiocyte monolayer after treatment with glycochenodeoxycholic acid. CONCLUSIONS: This bile duct-on-a-chip captured essential features of a simplified bile duct in structure and organ-level functions and represents an in vitro platform to study the pathophysiology of the bile duct using cholangiocytes from a variety of sources.

Coordinated development of the mouse extrahepatic bile duct: Implications for neonatal susceptibility to biliary injury.

BACKGROUND & AIMS: The extrahepatic bile duct is the primary tissue initially affected by biliary atresia. Biliary atresia is a cholangiopathy which exclusively affects neonates. Current animal models suggest that the developing bile duct is uniquely susceptible to damage. In this study, we aimed to define the anatomical and functional differences between the neonatal and adult mouse extrahepatic bile ducts. METHODS: We studied mouse passaged cholangiocytes, mouse BALB/c neonatal and adult primary cholangiocytes, as well as isolated extrahepatic bile ducts, and a collagen reporter mouse. The methods used included transmission electron microscopy, lectin staining, immunostaining, rhodamine uptake assays, bile acid toxicity assays, and in vitro modeling of the matrix. RESULTS: The cholangiocyte monolayer of the neonatal extrahepatic bile duct was immature, lacking the uniform apical glycocalyx and mature cell-cell junctions typical of adult cholangiocytes. Functional studies showed that the glycocalyx protected against bile acid injury and that neonatal cholangiocyte monolayers were more permeable than adult monolayers. In adult ducts, the submucosal space was filled with collagen I, elastin, hyaluronic acid, and proteoglycans. In contrast, the neonatal submucosa had little collagen I and elastin, although both increased rapidly after birth. In vitro modeling of the matrix suggested that the composition of the neonatal submucosa relative to the adult submucosa led to increased diffusion of bile. A Col-GFP reporter mouse showed that cells in the neonatal but not adult submucosa were actively producing collagen. CONCLUSION: We identified 4 key differences between the neonatal and adult extrahepatic bile duct. We showed that these features may have functional implications, suggesting the neonatal extrahepatic bile ducts are particularly susceptible to injury and fibrosis. LAY SUMMARY: Biliary atresia is a disease that affects newborns and is characterized by extrahepatic bile duct injury and obstruction, resulting in liver injury. We identify 4 key differences between the epithelial and submucosal layers of the neonatal and adult extrahepatic bile duct and show that these may render the neonatal duct particularly susceptible to injury.

G protein-coupled receptor gpr34l mutation affects thrombocyte function in zebrafish.

Haemostasis is a defence mechanism that has evolved to protect organisms from losing their circulating fluid. We have previously introduced zebrafish as a model to study the genetics of haemostasis to identify novel genes that play a role in haemostasis. Here, we identify a zebrafish mutant that showed prolonged time to occlusion (TTO) in the laser injury venous thrombosis assay. By linkage analysis and fine mapping, we found a mutation in the orphan G protein-coupled receptor 34 like gene (gpr34l) causing a change of Val to Glu in the third external loop of Gpr34l. We have shown that injection of zebrafish gpr34l RNA rescues the prolonged TTO defect. The thrombocytes from the mutant showed elevated levels of cAMP that supports the defective thrombocyte function. We also have demonstrated that knockdown of this gene by intravenous Vivo-Morpholino injections yielded a phenotype similar to the gpr34l mutation. These results suggest that the lack of functional Gpr34l leads to increased cAMP levels that result in defective thrombocyte aggregation.

Intraflagellar transport proteins are involved in thrombocyte filopodia formation and secretion.

Intraflagellar transport (IFT) proteins are vital for the genesis and maintenance of cilia. Our identification of ift122 transcripts in zebrafish thrombocytes that lack primary cilia was unexpected. IFT proteins serve transport in cilia, whose narrow dimensions may have necessitated the evolution of IFT from vesicular transport in ancestral eukaryotes. We hypothesized that IFTs might also facilitate transport within the filopodia that form when thrombocytes are activated. To test this possibility, we knocked down ift122 expression by injecting antisense Morpholino oligonucleotides (MOs) into zebrafish embryos. Laser-induced arterial thrombosis showed prolonged time to occlusion (TTO) of the vessel, as would be expected with defective thrombocyte function. Acute effects in adult zebrafish were evaluated by Vivo-Morpholino (Vivo-MO) knockdown of ift122. Vivo-MO morphants showed a prolonged time to thrombocyte aggregation (TTA) in the plate tilt assay after thrombocyte activation by the following agonists: ADP, collagen, PAR1 peptide, and epinephrine. A luminescence assay for ATP revealed that ATP secretion by thrombocytes was reduced in collagen-activated blood of Vivo-MO ift122 morphants. Moreover, DiI-C18 labeled morphant thrombocytes exposed to collagen showed reductions in filopodia number and length. Analysis of ift mutants, in which cilia defects have been noted, also showed prolongation of TTO in our arterial laser thrombosis assay. Additionally, collagen activation of wild-type thrombocytes led to a concentration of IFT122 both within and at the base of filopodia. Taken together these results, suggest that IFT proteins are involved in both the extension of filopodia and secretion of ATP, which are critical in thrombocyte function.

Knockdown of αIIb by RNA degradation by delivering deoxyoligonucleotides piggybacked with control vivo-morpholinos into zebrafish thrombocytes.

Morpholino and vivo-morpholino gene knockdown methods have been used to study thrombocyte function in zebrafish. However, a large-scale knockdown of the entire zebrafish genome using these technologies to study thrombocyte function is prohibitively expensive. We have developed an inexpensive gene knockdown method, which uses a hybrid of a control vivo-morpholino and a standard antisense oligonucleotide specific for a gene. This hybrid molecule is able to deliver antisense deoxyoligonucleotides into zebrafish thrombocytes because it piggybacks on a control vivo-morpholino. To validate use of this hybrid molecule in gene knockdowns, we targeted the thrombocyte specific αIIb gene with a hybrid of a control vivo-morpholino and an oligonucleotide antisense to αIIb mRNA. The use of this piggyback technology resulted in degradation of αIIb mRNA and led to thrombocyte functional defect. This piggyback method to knockdown genes is inexpensive since one control vivo-morpholino can be used to target many different genes by making many independent gene-specific oligonucleotide hybrids. Thus, this novel piggyback technology can be utilized for cost-effective large-scale knockdowns of genes to study thrombocyte function in zebrafish.

Role of hepsin in factor VII activation in zebrafish.

Factor VII, the initiator of the extrinsic coagulation cascade, circulates in human plasma mainly in its zymogen form, factor VII and in small amounts in its activated form, factor VIIa. However, the mechanism of initial generation of factor VIIa is not known despite intensive research using currently available model systems. Earlier findings suggested serine proteases factor VII activating protease and hepsin play a role in activating factor VII, however, it has remained controversial. In this paper we estimated the levels of factor VIIa and factor VII for the first time in zebrafish adult population and also reevaluated the role of the above two serine proteases in activating factor VII in vivo using zebrafish as a model system. Knockdown of factor VII activating protease and hepsin was performed followed by assaying for their effect on factor VIIa concentration and extrinsic coagulation as measured by the kinetic prothrombin time. Factor VII activating protease knockdown showed no change in kinetic prothrombin time and no effect on factor VIIa levels while hepsin knockdown increased the kinetic prothrombin time and significantly reduced the factor VIIa plasma levels. Our results thus indicate that hepsin plays a physiologically important role in factor VII activation and hemostasis in zebrafish.

Prostasin and hepatocyte growth factor B in factor VIIa generation: Serine protease knockdowns in zebrafish.

BACKGROUND: Blood clotting in humans is initiated by the binding of tissue factor to activated coagulation factor VII (FVIIa) in the plasma. Previous studies have reported that hepsin and factor VII (FVII)-activating protease are responsible for generating FVIIa. OBJECTIVES: We aimed to identify other proteases that may activate FVII using zebrafish as a model. METHODS: We screened 179 genes encoding serine protease domains using the piggyback knockdown method to identify genes involved in the activation of zebrafish Fvii. A prolonged kinetic prothrombin time (kPT) assay was used to detect gene knockdown effects. RESULTS: In the primary screen, 21 genes showed prolonged kPT. In the secondary screen, 14 of 21 genes showed positive results. In the tertiary screen, all 14 genes showed prolonged kPT. These 14 genes were knocked down again to estimate relative levels of zebrafish Fviia. Six genes, including known genes, such as f10 and novel prostasin and hepatocyte growth factor B (hgfb), showed lower Fviia levels. Fvii levels were affected only by the knockdown of f7 and not by the knockdown of the other five genes. CONCLUSIONS: Prostasin and hgfb are involved in generating Fviia. We hypothesize that prostasin exerts serine protease activity directly or indirectly to activate Fvii. As Hgfb has a mutated serine protease domain, it may not cleave Fvii but may bind to Fvii to induce autoactivation. The approach developed here may be extended to design other large-scale knockdown screens.

Distinct effects of different matrix proteoglycans on collagen fibrillogenesis and cell-mediated collagen reorganization.

The extracellular matrix (ECM) is a complex mixture composed of fibrillar collagens as well as additional protein and carbohydrate components. Proteoglycans (PGs) contribute to the heterogeneity of the ECM and play an important role in its structure and function. While the small leucine rich proteoglycans (SLRPs), including decorin and lumican, have been studied extensively as mediators of collagen fibrillogenesis and organization, the function of large matrix PGs in collagen matrices is less well known. In this study, we showed that different matrix PGs have distinct roles in regulating collagen behaviors. We found that versican, a large chondroitin sulfate PG, promotes collagen fibrillogenesis in a turbidity assay and upregulates cell-mediated collagen compaction and reorganization, whereas aggrecan, a structurally-similar large PG, has different and often opposing effects on collagen. Compared to versican, decorin and lumican also have distinct functions in regulating collagen behaviors. The different ways in which matrix PGs interact with collagen have important implications for understanding the role of the ECM in diseases such as fibrosis and cancer, and suggest that matrix PGs are potential therapeutic targets.

fMRI reliability in subjects with stroke.

Functional MRI (fMRI) has become one of the most commonly used neuroimaging tools to assess the cortical effects associated with rehabilitation, learning, or disease recovery in subjects with stroke. Despite this, there has been no systematic study of the reliability of the fMR signal in this population. The purpose of this study was to examine the within- and between-session reliability of fMRI in cortical and cerebellar structures in subjects with stroke during a complex, continuous visual motor task performed with the less affected hand. Nine subjects with stroke underwent four testing trials during two sessions separated by three weeks. Subjects performed a drawing task using an MRI compatible joystick while in the MRI. Methods of analysis evaluated included: percent signal intensity change, active voxel count and a voxel by voxel stat value analysis within and between testing sessions. Reliability was determined with Interclass correlation coefficients (ICC) in the following regions of interest: primary motor (M1), primary sensory (S1), premotor cortex (PMC), medial cerebellum (MCB), and lateral cerebellum (LCB). Results indicate that intensity change has superior reliability to the other methods of analysis (Average ICC across brain regions and trials: intensity change: 0.73, voxel count: 0.58, voxel by voxel: 0.67) and that generally with any analysis method, within-session reliability was higher than between-session, as indicated by higher ICC values across brain regions. Overall, when comparing between-session results, moderate to good reliability was obtained with intensity change (ICC: M1: 0.52, S1: 0.80, SMA: 0.78, PMC: 0.94, MCB: 0.86, and LCB: 0.59). These results show good reliability in subjects with stroke when performing a continuous motor task. These findings give confidence for interpreting fMRI test/retest research in subjects with stroke.

Neural substrates for motor imagery in severe hemiparesis.

BACKGROUND: The beneficial effects of imagined movements on motor learning and performance suggest that motor imagery is functionally close to preparatory and executive motor processes. OBJECTIVE: The purpose of this study was to examine the cortical processes associated with imagery of movement of the wrist in subjects with severe hemiparesis. METHODS: During fMRI, subjects with stroke performed alternating blocks of imagining wrist-tracking movements with the hemiparetic hand, active wrist-tracking movements with the unaffected hand, and resting. Control subjects performed the same tasks using an assigned hand. Cortical activation in the primary motor (M1), primary sensory (S1), supplementary motor area (SMA), and pre-SMA regions was determined through a laterality index of active voxels and signal intensity. Ability to imagine was assessed with an Imagery Rating Scale. RESULTS: All subjects displayed primarily contralateral control during the track condition. Healthy subjects demonstrated contralateral control in all areas during the imagine condition, whereas subjects with stroke displayed primarily contralateral activation in S1 but ipsilateral in M1 and SMA. The percentage change in signal intensity was greater in the ipsilateral hemisphere in subjects with stroke than in the ipsilateral hemisphere in healthy subjects during the imagine condition. Additionally, subjects with self-reported low ability to imagine displayed no difference in activation compared to those with high imagery ability. CONCLUSIONS: These findings are consistent with other works demonstrating primarily ipsilateral control of the hemiparetic hand in subjects with functional movement and lay the groundwork for further investigation into the ability of mental imagery to affect functionally relevant cortical control in subjects recovering from stroke.

Zebrafish thrombocytes: functions and origins.

Platelets play an important role in mammalian hemostasis. Thrombocytes of early vertebrates are functionally equivalent to mammalian platelets. A substantial amount of research has been done to study platelet function in humans as well as in animal models. However, to date only limited functional genomic studies of platelets have been performed but are low throughput and are not cost-effective. Keeping this in mind we introduced zebrafish, a vertebrate genetic model to study platelet function. We characterized zebrafish thrombocytes and established functional assays study not only their hemostatic function but to also their production. We identified a few genes which play a role in their function and production. Since we introduced the zebrafish model for the study of hemostasis and thrombosis, other groups have adapted this model to study genes that are associated with thrombocyte function and a few novel genes have also been identified. Furthermore, transgenic zebrafish with GFP-tagged thrombocytes have been developed which helped to study the production of thrombocytes and their precursors as well as their functional roles not only in hemostasis but also hematopoiesis. This paper integrates the information available on zebrafish thrombocyte function and its formation.