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, 158 (4), 1239-44

Proliferating Bile Duct Epithelial Cells Are a Major Source of Connective Tissue Growth Factor in Rat Biliary Fibrosis

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Proliferating Bile Duct Epithelial Cells Are a Major Source of Connective Tissue Growth Factor in Rat Biliary Fibrosis

N Sedlaczek et al. Am J Pathol.

Abstract

Connective tissue growth factor (CTGF) is a downstream mediator of transforming growth factor-beta1 (TGF-beta1) and thus a potential target for antifibrotic treatment strategies. CTGF is up-regulated in disorders such as atherosclerosis, scleroderma, and fibrosis of kidneys and lungs. We investigated the temporospatial expression patterns of CTGF and TGF-beta1 mRNA in rat livers with acute fibrogenesis (after a single dose of CCl(4)) and with advanced fibrosis (6 weeks after complete bile duct occlusion). Multiprobe ribonuclease protection assay revealed increasing TGF-beta1 and CTGF mRNA levels 6 hours after injection of CCl(4), with peak levels after 72 hours. In biliary fibrosis TGF-beta1 and CTGF mRNA levels increased fourfold and sevenfold, respectively (P: < 0.001). In situ hybridization combined with cell-specific markers revealed CTGF transcripts in desmin-positive cells after a single dose of carbon tetrachloride, whereas no transcripts were found in normal livers. In biliary fibrosis, however, proliferating bile duct epithelial cells were the predominant source of CTGF mRNA. We conclude that in rat liver fibrogenesis CTGF is up-regulated in close association with TGF-beta1 and that, contrary to a previous report, not solely hepatic stellate cells but activated bile duct epithelial cells are the main source of this profibrogenic factor.

Figures

Figure 1.
Figure 1.
Coordinate expression of CTGF and TGF-β1 in acute liver fibrogenesis. A: Multiprobe ribonuclease protection assay for CTGF and TGF-β1 mRNA in rats after acute CCl4-intoxication. Twenty μg of liver RNA were hybridized with [α-32P]-labeled CTGF and TGF-β1 riboprobes. After RNase T1 digestion the protected probes were resolved through denaturing polyacrylamide gel electrophoresis and subjected to autoradiography at −70°C for 16 hours. B: P, full-length probes; T, tRNA control. Autoradiographic signals were normalized to that of GAPDH and expressed as arbitrary units. Each time point represents the mean value of three rats. *, P < 0.05 compared with all other time points.
Figure 2.
Figure 2.
Coordinate expression of CTGF and TGF-β1 in chronic liver fibrogenesis. Multiprobe ribonuclease protection assay for CTGF and TGF-β1 mRNA from rats after BDO for 6 weeks. Twenty μg of liver RNA were hybridized with [α-32P]-labeled CTGF and TGF-β1 riboprobes and processed as described in Figure 1 ▶ . P, full-length probes; T, tRNA control. Error bars represent standard errors derived from five rats. *, P < 0.001 compared with sham-operated controls.
Figure 3.
Figure 3.
Expression patterns of CTGF and procollagen α1(I) mRNA in acute and chronic hepatic fibrogenesis. Radioactive in situ hybridization, in part in combination with immunostaining for desmin (A-D, G, and H) or pan-cytokeratin (E) on rat livers. CTGF antisense: A: Normal liver portal tract; labeling of HSCs/portal fibroblasts (original magnification, ×600). B: Six hours after CCl4, labeling of desmin-positive HSC and endothelial cells (original magnification, ×400). C: Six hours after CCl4, prominent signals in hepatic arterial (ha) myocytes in a larger portal tract, no expression over a larger bile duct (bd) (original magnification, ×200). D: Seventy-two hours after CCl4, strong labeling of single centrilobular HSC and endothelial cells of a terminal hepatic vein (thv) (original magnification, ×600). E: Seventy-two hours after CCl4, enhanced CTGF mRNA expression in most portal tract fibroblasts (original magnification, ×400). F and G: CTGF mRNA is almost exclusively expressed in desmin-negative (F: original magnification, ×900) and pan-cytokeratin-positive (G: original magnification, ×600) bile duct epithelia (bd in biliary fibrosis); note the patchy ductular CTGF mRNA expression pattern. H: Up-regulation of procollagen α1(I) mRNA in desmin-positive myofibroblasts surrounding proliferating bile ducts (bd) after 6 weeks of BDO (original magnification, ×700).

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