Background & Aims Vascular endothelial growth factor (VEGF)—induced angiogenesis is implicated in fibrogenesis and portal hypertension. However, the function of VEGF in fibrosis resolution has not been explored. Methods We developed a cholecystojejunostomy procedure to reconstruct biliary flow after bile duct ligation in C57BL/6 mice to generate a model of fibrosis resolution. These mice were then given injections of VEGF-neutralizing (mcr84) or control antibodies, and other mice received an adenovirus that expressed mouse VEGF or a control vector. The procedure was also performed on macro-phage fas-induced apoptosis mice, in which macrophages can be selectively depleted. Liver and blood samples were collected and analyzed in immunohistochemical, morphometric, vascular permeability, real-time polymerase chain reaction, and flow cytometry assays. Results VEGF-neutralizing antibodies prevented development of fibrosis but also disrupted hepatic tissue repair and fibrosis resolution. During fibrosis resolution, VEGF inhibition impaired liver sinusoidal permeability, which was associated with reduced monocyte migration, adhesion, and infiltration of fibrotic liver. Scar-associated macrophages contributed to this process by producing the chemokine (C-X-C motif) ligand 9 and matrix metalloproteinase 13. Resolution of fibrosis was impaired in macrophage fas-induced apoptosis mice but increased after overexpression of chemokine (C-X-C motif) ligand 9. Conclusions In a mouse model of liver fibrosis resolution, VEGF promoted fibrogenesis, but was also required for hepatic tissue repair and fibrosis resolution. We observed that VEGF regulates vascular permeability, monocyte infiltration, and scar-associated macrophages function.

Primary cilia are multisensory organelles recently found to be absent in some tumor cells, but the mechanisms of deciliation and the role of cilia in tumor biology remain unclear. Cholangiocytes, the epithelial cells lining the biliary tree, normally express primary cilia and their interaction with bile components regulates multiple processes, including proliferation and transport. Utilizing cholangiocarcinoma (CCA) as a model, we found primary cilia are reduced in CCA by a mechanism involving histone deacetylase 6 (HDAC6). The experimental deciliation of normal cholangiocyte cells increased the proliferation rate and induced anchorage-independent growth. Furthermore, deciliation induced the activation of MAPK and Hedgehog signaling, two important pathways involved in CCA development. We found HDAC6 is overexpressed in CCA and overexpression of HDAC6 in normal cholangiocytes induced deciliation, and increased both proliferation and anchorage-independent growth. To evaluate the effect of cilia restoration on tumor cells, we targeted HDAC6 by shRNA or by the pharmacologic inhibitor, tubastatin-A. Both approaches restored the expression of primary cilia in CCA cell lines and decreased cell proliferation and anchorage-independent growth. The effects of tubastatin-A were abolished when CCA cells were rendered unable to regenerate cilia by stable transfection of IFT88-shRNA. Finally, inhibition of HDAC6 by tubastatin-A also induced a significant decrease in tumor growth in a CCA animal model. Our data support a key role for primary cilia in malignant transformation, provide a plausible mechanism for their involvement, and suggest that restoration of primary cilia in tumor cells by HDAC6 targeting may be a potential therapeutic approach for CCA.

MicroRNAs, central players of numerous cellular processes, regulate mRNA stability or translational efficiency. Although these molecular events are established, the mechanisms regulating microRNA function and expression remain largely unknown. The microRNA let-7i regulates Toll-like receptor 4 expression. Here, we identify a novel transcriptional mechanism induced by the protozoan parasite Cryptosporidium parvum and Gram(؊) bacteria-derived lipopolysaccharide (LPS) mediating let-7i promoter silencing in human biliary epithelial cells (cholangiocytes). Using cultured cholangiocytes, we show that microbial stimulus decreased let-7i expression, and promoter activity. Analysis of the mechanism revealed that microbial infection promotes the formation of a NFB p50-C/EBP␤ silencer complex in the regulatory sequence. Chromatin immunoprecipitation assays (ChIP) demonstrated that the repressor complex binds to the let-7i promoter following microbial stimulus and promotes histone-H3 deacetylation. Our results provide a novel mechanism of transcriptional regulation of cholangiocyte let-7i expression following microbial insult, a process with potential implications for epithelial innate immune responses in general.Intrahepatic bile ducts constitute a complex 3-dimensional tubular network, the biliary tract, through which bile is transported to the duodenum. Human bile is sterile under normal physiological conditions (1); however, the biliary tract is periodically exposed to pathogens, including Escherichia coli and the protozoan parasite Cryptosporidium parvum, or pathogenderived molecules, including Gram-negative bacteria-derived lipopolysaccharide (LPS).2 Upon pathogen recognition, a phenotypic transition occurs through which biliary epithelial cells (cholangiocytes) promote the innate and adaptive immune responses (1-4). Indeed, cholangiocytes express a variety of pathogen recognition receptors and actively participate in the innate immune response through the secretion of cytokines/ chemokines (5, 6), expression of adhesion molecules (7-9), and antimicrobial peptides (1, 10). Expression of these immuneassociated genes is a highly regulated process to assure that the epithelium recognizes and responds to invading pathogens, but does not induce injury through an inappropriate immune response. Recent reports suggest the microRNA machinery contributes to the regulation of the immune-associated gene expression. MicroRNAs are small (21-23 nt) RNA molecules that target and regulate the stability or translational efficiency of mRNAs (11). These regulatory RNAs are transcribed as mono-or polycistronic primary microRNAs (pri-microRNAs), which are sequentially processed to precursor and the functionally active mature microRNA. The molecular mechanisms regulating the expression of most microRNAs remain largely unknown.Using a human cholangiocyte cell culture model of biliary cryptosporidiosis, we previously reported that let-7i, a member of the let-7 family of microRNAs, targets Toll-like receptor 4 mRNA and limits the expression of...