Jonathan A. Dranoff

Transforming growth factor‐β and substrate stiffness regulate portal fibroblast activation in culture

Myofibroblasts derived from portal fibroblasts are important fibrogenic cells in the early stages of biliary fibrosis. In contrast to hepatic stellate cells, portal fibroblasts have not been well studied in vitro, and little is known about their myofibroblastic differentiation. In this article we report the isolation and characterization of rat portal fibroblasts in culture. We demonstrate that primary portal fibroblasts undergo differentiation to α‐smooth muscle actin–expressing myofibroblasts over 10–14 days. Marker analysis comparing portal fibroblasts to hepatic stellate cells demonstrated that these are distinct populations and that staining with elastin and desmin can differentiate between them. Portal fibroblasts expressed elastin at all stages in culture but never expressed desmin, whereas hepatic stellate cells consistently expressed desmin but never elastin. Immunostaining of rat liver tissue confirmed these results in vivo. Characterization of portal fibroblast differentiation in culture demonstrated that these cells required transforming growth factor‐β (TGF‐β): cells remained quiescent in the presence of a TGF‐β receptor kinase inhibitor, whereas exogenous TGF‐β1 enhanced portal fibroblast α‐smooth muscle actin expression and stress fiber formation. In contrast, platelet‐derived growth factor inhibited myofibroblastic differentiation. Portal fibroblasts were also dependent on mechanical tension for myofibroblastic differentiation, and cells cultured on polyacrylamide supports of variable stiffness demonstrated an increasingly myofibroblastic phenotype as stiffness increased. Conclusion: Portal fibroblasts are morphologically and functionally distinct from hepatic stellate cells. Portal fibroblast myofibroblastic differentiation can be modeled in culture and requires both TGF‐β and mechanical tension. (HEPATOLOGY 2007.)

Portal fibroblasts: Underappreciated mediators of biliary fibrosis†

Portal fibroblasts are an important yet often overlooked nonparenchymal cell population in the liver. They are distinct from hepatic stellate cells, yet like stellate cells differentiate in the setting of chronic injury to fibrogenic myofibroblasts, playing an important role in collagen production in the fibrotic liver. Portal fibroblasts (PFs) are located adjacent to bile duct epithelia and thus play a particularly significant role in biliary fibrosis. New data suggest that they may also have key functions independent of fibrogenesis. This review addresses the definition and characteristics of PFs as well as their signaling pathways, interactions with the biliary epithelium, and contributions to liver pathobiology. Conclusion: PFs are an important and multifunctional nonparenchymal cell population in need of further study. (HEPATOLOGY 2010.)

Portal fibroblasts regulate the proliferation of bile duct epithelia via expression of NTPDase2

Bile duct epithelia are the target of a number of “cholangiopathies” characterized by disordered bile ductular proliferation. Although mechanisms for bile ductular proliferation are unknown, recent evidence suggests that extracellular nucleotides regulate cell proliferation via activation of P2Y receptors. Portal fibroblasts may regulate bile duct epithelial P2Y receptors via expression of the ecto-nucleotidase NTPDase2. Thus, we tested the hypothesis that portal fibroblasts regulate bile duct epithelial proliferation via expression of NTPDase2. We generated a novel co-culture model of Mz-ChA-1 human cholangiocarcinoma cells and primary portal fibroblasts. Cell proliferation was measured by bromodeoxyuridine uptake. NTPDase2 expression was assessed by immunofluorescence and quantitative real-time reverse transcription PCR. NTPDase2 expression in portal fibroblasts was blocked using short interfering RNA. NTPDase2 overexpression in portal myofibroblasts isolated from bile duct-ligated rats was achieved by cDNA transfection. Co-culture of Mz-ChA-1 cells with portal fibroblasts decreased their proliferation to 26% of control. Similar decreases in Mz-ChA-1 proliferation were induced by the soluble ecto-nucleotidase apyrase and the P2 receptor inhibitor suramin. The proliferation of Mz-ChA-1 cells returned to baseline when NTPDase2 expression in portal fibroblasts was inhibited using NTPDase2-specific short interfering RNA. Untransfected portal myofibroblasts lacking NTPDase2 had no effect on Mz-ChA-1 proliferation, yet portal myofibroblasts transfected with NTPDase2 cDNA inhibited Mz-ChA-1 proliferation. We conclude that portal fibroblasts inhibit bile ductular proliferation via expression of NTPDase2 and blockade of P2Y activation. Loss of NTPDase2 may mediate the bile ductular proliferation typical of obstructive cholestasis. This novel cross-talk signaling pathway may mediate pathologic alterations in bile ductular proliferation in other cholangiopathic conditions.

Autocrine release of TGF-β by portal fibroblasts regulates cell growth

Portal fibroblasts (PF) are a newly isolated population of fibrogenic cells in the liver postulated to play a significant role in early biliary fibrosis. Because transforming growth factor‐β (TGF)‐β is a key growth factor in fibrosis, we characterized the response of PF to TGF‐β. We demonstrate that PF produce significant amounts of TGF‐β2 and, unlike activated hepatic stellate cells (HSC), express all three TGF‐β receptors and are growth inhibited by TGF‐β1 and TGF‐β2. Fibroblast growth factor (FGF)‐2, but not platelet derived growth factor (PDGF), causes PF proliferation. These data suggest a mechanism whereby HSC eclipse PF as the dominant myofibroblast population in biliary fibrosis.

Adenosine induces loss of actin stress fibers and inhibits contraction in hepatic stellate cells via Rho inhibition

The Rho/ROCK pathway is activated in differentiated hepatic stellate cells (HSCs) and is necessary for assembly of actin stress fibers, contractility, and chemotaxis. Despite the importance of this pathway in HSC biology, physiological inhibitors of the Rho/ROCK pathway in HSCs are not known. We demonstrate that adenosine induces loss of actin stress fibers in the LX‐2 cell line and primary HSCs in a manner indistinguishable from Rho/ROCK inhibition. Loss of actin stress fibers occurs via the A2a receptor at adenosine concentrations above 10 μM, which are present during tissue injury. We further demonstrate that loss of actin stress fibers is due to a cyclic adenosine monophosphate, protein kinase A–mediated pathway that results in Rho inhibition. Furthermore, a constitutively active Rho construct can inhibit the ability of adenosine to induce loss of actin stress fibers. Actin stress fibers are required for HSC contraction, and we demonstrate that adenosine inhibits endothelin‐1 and lysophosphatidic acid–mediated HSC contraction. We propose that adenosine is a physiological inhibitor of the Rho pathway in HSCs with functional consequences, including loss of HSC contraction. (HEPATOLOGY 2009;49:185‐194)

Isolation of Primary Rat Liver Fibroblasts

Introduction One of the major advances in liver research in the past decade was the ability to isolate distinct liver cell populations. Although there are established methods of isolating hepatocytes, cholangiocytes, and stellate cells, before this study no technique for liver fibroblast isolation had been devised. Consequently, we developed a technique to isolate primary rat liver fibroblasts. Methods Fibroblasts were isolated from a freshly perfused rat liver with a modification of the procedure for isolation of rat cholangiocytes. Cell markers were assessed with the use of confocal immunofluorescence. Cell morphology was assessed with transmission electron microscopy. Expression of procollagen-1 was assessed by reverse transcription polymerase chain reaction. Results The appearance of cells with fibroblast morphology was first noted at 48 hours, and almost all cells in culture had fibroblast morphology at 96 hours. Putative fibroblasts stained for vimentin, but not for smooth muscle actin, von Willebrand factor, or cytokeratins. Cell morphology was consistent with that of fibroblasts and showed no features of epithelial, endothelial, or smooth muscle cells. Liver fibroblasts expressed procollagen-1 mRNA. Conclusion Primary isolated rat fibroblasts can be produced from a freshly perfused rat liver with a modification of standard cell culture methods. The role of fibroblasts in liver physiology can now be studied directly.

Advances in cholangiocyte immunobiology

Cholangiocytes, or bile duct epithelia, were once thought to be the simple lining of the conduit system comprising the intra- and extrahepatic bile ducts. Growing experimental evidence demonstrated that cholangiocytes are in fact the first line of defense of the biliary system against foreign substances. Experimental advances in recent years have unveiled previously unknown roles of cholangiocytes in both innate and adaptive immune responses. Cholangiocytes can release inflammatory modulators in a regulated fashion. Moreover, they express specialized pattern-recognizing molecules that identify microbial components and activate intracellular signaling cascades leading to a variety of downstream responses. The cytokines secreted by cholangiocytes, in conjunction with the adhesion molecules expressed on their surface, play a role in recruitment, localization, and modulation of immune responses in the liver and biliary tract. Cholangiocyte survival and function is further modulated by cytokines and inflammatory mediators secreted by immune cells and cholangiocytes themselves. Because cholangiocytes act as professional APCs via expression of major histocompatibility complex antigens and secrete antimicrobial peptides in bile, their role in response to biliary infection is critical. Finally, because cholangiocytes release mediators critical to myofibroblastic differentiation of portal fibroblasts and hepatic stellate cells, cholangiocytes may be essential in the pathogenesis of biliary cirrhosis.

Transnasal endoscopy for enteral feeding tube placement in critically ill patients

Objective:Early enteral feedings may improve outcomes in critically ill patients. Recently, transnasal endoscopy with an ultrathin transnasal endoscope has been shown to be of value for diagnostic endoscopy without conscious sedation. We developed a technique for the placement of postpyloric feeding tubes in critically ill patients using transnasal endoscopy. We describe our initial experience in a consecutive series of patients.Methods:We collected data on consecutive intensive care unit patients undergoing bedside transnasal endoscopy for nasoenteric feeding tube placement using a standardized technique. Tube position was confirmed in all patients with a plain abdominal radiograph. Tube placement was deemed successful if the feeding tube traversed the pylorus.Results:Transnasal endoscopy was completed in all fourteen patients, as was placement of a feeding tube. Feeding tubes were successfully placed in the jejunum or duodenum in 13 of the 14 patients (93%). Tubes remained in place from 3 to 45 days (mean 16 days). Two patients required conscious sedation during tube placement, and two ultimately required percutaneous gastrostomy.Conclusion:Transnasal endoscopy allows simple and successful postpyloric feeding tube placement at the bedside of critically ill patients. This method can facilitate early enteral feeding in intensive care units.

The Spatial Distribution of Inositol 1,4,5-Trisphosphate Receptor Isoforms Shapes Ca2+ Waves

Cytosolic Ca2+ is a versatile second messenger that can regulate multiple cellular processes simultaneously. This is accomplished in part through Ca2+ waves and other spatial patterns of Ca2+ signals. To investigate the mechanism responsible for the formation of Ca2+ waves, we examined the role of inositol 1,4,5-trisphosphate receptor (InsP3R) isoforms in Ca2+ wave formation. Ca2+ signals were examined in hepatocytes, which express the type I and II InsP3R in a polarized fashion, and in AR4-2J cells, a nonpolarized cell line that expresses type I and II InsP3R in a ratio similar to what is found in hepatocytes but homogeneously throughout the cell. Expression of type I or II InsP3R was selectively suppressed by isoform-specific DNA antisense in an adenoviral delivery system, which was delivered to AR4-2J cells in culture and to hepatocytes in vivo. Loss of either isoform inhibited Ca2+ signals to a similar extent in AR4-2J cells. In contrast, loss of the basolateral type I InsP3R decreased the sensitivity of hepatocytes to vasopressin but had little effect on the initiation or spread of Ca2+ waves across hepatocytes. Loss of the apical type II isoform caused an even greater decrease in the sensitivity of hepatocytes to vasopressin and resulted in Ca2+ waves that were much slower and delayed in onset. These findings provide evidence that the apical concentration of type II InsP3Rs is essential for the formation of Ca2+ waves in hepatocytes. The subcellular distribution of InsP3R isoforms may critically determine the repertoire of spatial patterns of Ca2+ signals.

Contribution of Myofibroblasts of Different Origins to Liver Fibrosis

The most common cause of liver failure is cirrhosis, due to progressive liver fibrosis and other architectural changes in the liver. Fibrosis occurs after liver injury or stress and results directly from an imbalance between the processes of extracellular matrix synthesis (fibrogenesis) and degradation (fibrolysis). Although research studies have identified several promising targets at the molecular level, current therapies to prevent and treat hepatic fibrosis in patients have only shown limited success. It is well established that liver myofibroblasts (MFs) are the primary effector cells responsible for the extensive extracellular matrix accumulation and scar formation observed during hepatic fibrosis, in both clinical and experimental settings. Thus, as the major fibrogenic cells implicated in wound healing and tissue repair response, liver MFs could represent excellent targets for antifibrotic therapies. Still, the exact nature and identities of liver MFs precursors have yet to be resolved, and their relative contribution to hepatic fibrosis to be determined. The goal of this review is to examine the relative importance of liver MF precursors in the pathogenesis of liver fibrosis.