Connective tissue growth factor is involved in pancreatic repair and tissue remodeling in human and rat acute necrotizing pancreatitis

Abstract

Objective: To analyze the involvement of connective tissue growth factor (CTGF) in the transforming growth factor-beta (TGF-beta) pathway during acute necrotizing pancreatitis (ANP) in humans and rats.

Summary background data: Connective tissue growth factor is involved in several fibrotic diseases and has a critical role in fibrogenesis and tissue remodeling after injury.

Methods: Normal human pancreas tissue samples were obtained through an organ donor program from five individuals without a history of pancreatic disease. Human ANP tissues were obtained from eight persons undergoing surgery for this disease. In rats, ANP was induced by intraductal infusion of taurocholate. The expression of CTGF was studied by Northern blot analysis, in situ hybridization, and immunohistochemistry in both human and rat pancreatic tissue samples.

Results: Northern blot analysis revealed enhanced CTGF mRNA expression in human ANP tissue samples compared with normal controls. In addition, a concomitant increase in TGF-beta1 was present. By in situ hybridization, CTGF mRNA was localized in the remaining acinar and ductal cells and in fibroblasts. In regions of intense damage adjacent to areas of necrosis, CTGF mRNA signals were most intense. Inflammatory cells were devoid of any CTGF mRNA signals. By immunohistochemistry, CTGF protein was localized at high levels in the same cell types as CTGF mRNA. In ANP in rats, concomitantly enhanced mRNA levels of CTGF, TGF-beta1, and collagen type 1 were present, with a biphasic peak pattern on days 2 to 3 and day 7 after induction of ANP.

Conclusions: These data indicate that CTGF participates in tissue remodeling in ANP. The expression of CTGF predominantly in the remaining acinar and ductal cells indicates that extracellular matrix synthesis after necrosis is at least partly regulated by the remaining pancreatic parenchyma and only to a minor extent by inflammatory cells. Blockage of CTGF, a downstream mediator of TGF-beta in fibrogenesis, might be useful as a target to influence and reduce fibrogenesis in this disorder.

Figure 1. Northern blot analysis of connective tissue growth factor (CTGF) and transforming growth factor-β1 (TGF-β1) mRNA in normal human pancreas (lanes 1–4) and human acute necrotizing pancreatitis tissue samples (lanes 5–12). In the acute necrotizing pancreatitis samples, concomitantly enhanced CTGF and TGF-β1 mRNA expression levels were present compared with the normal controls. 7S RNA was used to assess equivalent RNA loading.

Figure 2. Northern blot analysis of connective tissue growth factor (CTGF), transforming growth factor-β1 (TGF-β1), TGF-β2, TGF-β3, collagen type 1, and amylase mRNA gene expression in control normal pancreas (NP; lanes 1 and 2) compared with rats with acute necrotizing pancreatitis (lanes 3–11). Concomitant high values of CTGF, TGF-β1, and collagen type 1 mRNA expression were present on days 2 to 3 and again on day 7. Amylase mRNA expression showed initially marked reduction with a progressive recovery. 7S RNA was used to assess equivalent RNA loading.

Figure 3. In situ hybridization of connective tissue growth factor (CTGF) mRNA expression in human tissue sections of normal pancreas (A) and acute necrotizing pancreatitis (B, C, D) samples. In acute necrotizing pancreatitis tissue sections, CTGF mRNA signals were mainly present in remaining acinar cells and in fibroblasts, especially in those areas adjacent to the necrosis (B, C). Inflammatory cells were devoid of CTGF mRNA signals (C, arrows). In situ hybridization experiments using the DIG-labeled sense probe corresponding to the antisense probe failed to produce hybridization signals (E). Original magnification ×200 (A–C); ×400 (D, E).

Figure 4. Immunohistochemical analysis of connective tissue growth factor (CTGF) in human tissue sections of normal pancreas (A) and acute necrotizing pancreatitis (B, C, D) samples. In acute necrotizing pancreatitis tissue sections, CTGF immunoreactivity was mainly present in the cells of the small ducts and in all remaining acinar cells, especially in those areas adjacent to the necrosis (B, C, D, arrows). i, islet; d = duct. Original magnification ×100 (A, B); ×200 (C); ×400 (D).