3D, three-dimensional; BDL, bile duct ligation;
CCl4, carbon tetrachloride; cDNA, complementary DNA; CO2, carbon dioxide; DAPI, 4′,6-diamidino-2-phenylindole; DN, dominant-negative; FGF, fibroblast growth factor; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; H&E, hematoxylin and eosin; Hb, hemoglobin; HUVEC, human umbilical vein endothelial cell; LEC, liver endothelial cell; LPS, lipopolysaccharide; Pim inhibitor MLEC, murine liver endothelial cell; MMP, matrix metalloproteinase; mRNA, messenger RNA; MT, mutant; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; MyD88, myeloid differentiation protein 88; PCR, polymerase chain reaction; RT-PCR, real-time polymerase chain reaction; SEM, standard error of the mean; siRNA, short interfering RNA; TLR, toll-like receptor; TRAM, toll-like receptor adaptor Cell Cycle inhibitor molecule; VEGF, vascular endothelial growth factor; vWF, von Willebrand factor; WT, wild-type; YFP, yellow fluorescent protein. C3H/HeOuJ [TLR4–wild-type (WT)] mice and C3H/HeJ [TLR4-mutant (MT)] mice, which carry a spontaneous mutation that confers a loss of TLR4 function, were purchased from Jackson Laboratories (Bar Harbor, ME). These animals have similar levels of tumor necrosis
factor-α under basal conditions but impaired production in response to LPS.13 Bile duct ligation (BDL) and sham surgeries were performed as previously described.14 For carbon tetrachloride (CCl4)–induced fibrosis studies, CCl4 (1 MCE公司 mg/kg of body weight) or vehicle (olive oil) was injected intraperitoneally for a period of 6 weeks as previously described.15 LECs were isolated from mice as previously described,16, 17 and the purity was assessed (supplementary Fig- 1). All procedures were approved by the Mayo Clinic Institutional Animal Care and Use Committee. Human LECs (ScienCell, San Diego, CA) were grown under standard tissue
culture conditions [a humidified 5% carbon dioxide (CO2) incubator at 37°C] in media containing 5% fetal bovine serum, 2% endothelial cell growth supplement, and 1% penicillin/streptomycin (ScienCell). Retroviral transduction and short interfering RNA (siRNA) transfection were performed as we previously described.18 Two distinct siRNAs for TLR4 within the coding regions starting at 105 and 174 bp and MyD88 were gifts from Steven P. O’Hara, whereas TRAM siRNA was commercially obtained (Thermo Scientific). Human MyD88 full-length and dominant-negative N-terminal truncation MT constructs were polymerase chain reaction (PCR)–amplified from pUNO-hMyD88 and pDeNy-hMyD88 (InvivoGen), respectively, and the amplified fragments were subcloned into the pMMP retroviral vector.