Fibrogenic signals in patients with radiation enteritis are associated with increased connective tissue growth factor expression

Purpose: To investigate the expression of a new fibrogenic cytokine the connective tissue growth factor (CTGF) in intestinal radiation fibrosis and to characterize the mesenchymal cell subtypes involved in CTGF synthesis and collagen deposition. Methods and Materials: Sixteen patients with radiation enteritis that occurred after radiotherapy for pelvic malignancies and 6 with histologically normal bowel entered the study. Immunohistochemistry, Western blot analysis, and real-time reverse transcriptase-polymerase chain reaction were performed to study CTGF expression, along with other known markers of radiation fibrosis: the pro-fibrogenic cytokine transforming growth factor (TGF)-β1 and phenotypic markers of the fibroblast differentiation the α-sm actin (A), vimentin (V), and desmin (D). Finally, the collagen accumulation was measured by Sirius red staining and colorimetric assay. Results: Radiation enteritis was characterized by increased collagen content within the intestinal wall. CTGF immunoreactivity, protein, and mRNA level were increased in radiation enteritis compared with the healthy bowel. On the contrary, no increase of the TGF-β1 mRNA level was observed in radiation enteritis compared with healthy bowel, and the level of TGF-β protein was slightly increased in radiation enteritis. A co-localization of CTGF immunoreactivity and collagen deposition was found in the extracellular matrix and subtypes of activated mesenchymal cells with a fibroblast phenotype (V⁺/D^-/A^-) and myofibroblast phenotype (V⁺/D^-/+/A⁺). Conclusion: The increased level of CTGF protein and mRNA associated with the accumulation of fibroblasts/myofibroblasts and collagen deposition were parts of the fibrogenic signals involved in the persistence of late intestinal radiation fibrosis.