Cellular Sources of Transforming Growth Factor-β Isoforms in Early and Chronic Radiation Enteropathy

Abstract

The three mammalian transforming growth factor (TGF)-beta isoforms (TGF-beta1, TGF-beta2, and TGF-beta3) differ in their putative roles in radiation-induced fibrosis in intestine and other organs. Furthermore, tissue specificity of TGF-beta action may result from temporal or spatial changes in production and/or activation. The present study examined shifts in the cell types expressing TGF-beta mRNA relative to TGF-beta immunoreactivity and histopathological injury during radiation enteropathy development. A 4-cm loop of rat small intestine was locally exposed to O, 12, or 21-Gy single doses of x-irradiation. Sham-irradiated and irradiated intestine were procured 2 and 26 weeks after irradiation. Cells expressing the TGF-beta1, TGF-beta2, or TGF-beta3 transcripts were identified by in situ hybridization with digoxigenin-labeled riboprobes. Intestinal wall TGF-beta immunoreactivity was measured using computerized image analysis, and structural radiation injury was assessed by quantitative histopathology. Normal intestinal epithelium expressed transcripts for all three TGF-beta isoforms. Two weeks after irradiation, regenerating crypts, inflammatory cells, smooth muscle cells, and mesothelium exhibited increased TGF-beta1 expression and, to a lesser degree, TGF-beta2 and TGF-beta3 expression. Twenty-six weeks after irradiation, TGF-beta2 and TGF-beta3 expression had returned to normal. In contrast, TGF-beta1 expression remained elevated in smooth muscle, mesothelium, endothelium, and fibroblasts in regions of chronic fibrosis. Extracellular matrix-associated TGF-beta1 immunoreactivity was significantly increased at both observation times, whereas, TGF-beta2 and TGF-beta3 immunoreactivity exhibited minimal postradiation changes. Intestinal radiation injury is associated with overexpression of all three TGF-beta isoforms in regenerating epithelium. Radiation enteropathy was also associated with sustained shifts in the cellular sources of TGF-beta1 from epithelial cells to cells involved in the pathogenesis of chronic fibrosis. TGF-beta2 and TGF-beta3 did not exhibit consistent long-term changes. TGF-beta1 appears to be the predominant isoform in radiation enteropathy and may be more important in the mechanisms of chronicity than TGF-beta2 and TGF-beta3.

Essential Role of Plasminogen Activator Inhibitor Type-1 in Radiation Enteropathy

“…Intestinal radiation injury is associated with increased TGF-␤1 expression. 3 We show that PAI-1 is overexpressed in the endothelium of pathological tissue from patients treated with radiotherapy. Interestingly, radiation-induced damage in patients was associated with an increase in phosphorylated Smad 2/3 in the endothelium, suggesting that the Smad pathway may be involved in the overexpression of PAI-1 in ECs in vivo.…”

“…Slides were stained with hematoxylin-eosin-saffron and Sirius red. Radiation injury was determined using a described and validated radiation injury scoring system 3,4 administered independently by two authors in a blinded manner, and discrepancies were resolved by discussion. CD45 immunostaining was performed using rat antimouse common leukocyte antigen (BD Bioscience, Erembodegem, Belgium).…”

“…Among these factors, transforming growth factor (TGF)-␤1 is considered a key fibrogenic cytokine involved in radiation-induced intestinal damage. 3,4 TGF-␤1 up-regulates expression of several molecules that could contribute to acute and late radiation damage. Among them, we hypothesized that plasminogen activator inhibitor type-1 (PAI-1) is a critical mediator that triggers pathways leading to acute and late normal tissue lesions after irradiation.…”

Current concepts in radiation enteritis and implications for future clinical trials

“…All three isoforms are overexpressed in the early postradiation phase; however, by 26 weeks after radiation, only isoform ␤1 remains elevated. 17 In the first 2 weeks after radiation, TGF-␤1 messenger RNA is increased in epithelial cells, fibroblasts of submucosa and subserosa, and smooth muscles of the intestinal wall. However, at 26 weeks, the expression of TGF-␤1 in epithelial cells has returned to baseline and remains elevated in vascular endothelial cells, fibroblasts, and smooth muscles cells.…”

“…78 In another study with the same dose and frequency of IFN-␥, there also was a stabilization of visceral involvement. 79 A higher dose of IFN-␥ (150 mcg per day) induced a significant decrease in skin thickness compared with a lower dose (60 mcg per 17 Rat Intestines 0, 12 ISH Increased Wang et al 18 Rat Intestines 0, 12 CIA, IH Increased Hauer-Jensen et al 19 Rat Intestines 21 RTP, ISH Increased Zheng et al 20 Mouse Intestines 21 RTP, IH Inhibited by soluble TGF-␤ receptor Richter et al 23 Human Intestines NS IH Increased Bai et al 38 Rat Lungs 30 IH, ISH Increased Rube et al 39 Mouse Lungs 6, 12 IH, RTP Increased Rubin et al 43 Mouse Lungs 5 IH, ISH Increased Anscher et al 45 Human Lungs NS EIA Increased Randall and Coggle 50 Mouse Skin 50 PCR Increased Martin et al 51 Pig Skin 140 NBH, SBT, IF Increased Lefaix et al 55 Pig Skin 160 US, IH Inhibited by PTX and vit E Zhao et al 60 Rat Kidney 1-10 NBA Increased Robbins et al 61 Rat Kidney 10, 15, 20 IH Increased Zhao et al 62 Rat Kidney 1-10 WBA, LA Increased Datta et al 63 Rat Kidney 15 EIA Increased Seong et al 65 Rat Liver 25 IH Increased Geraci and Mariqno 66 Rat Liver 25 RH Increased Anscher et al 67 Rat Liver NS IH Increased TGF-␤: transforming growth factor beta; Gy: Gray; NS: not specified; IH: immunohistochemistry; RIS: radiation injury scoring; TBS: toluidine blue stain; ISH: in situ hybridization; CIA: computerized image analysis; RTP: reverse transcriptase polymerase; EIA: enzyme-linked immunosorbent assay; US: ultrasound; NBH: Northern blot hybridization; SBH: Southern blot hybridization; PCR: polymerase chain reaction; NBA: Northern blot analysis; WBA: Western blot analysis; LA: luciferase assay; RH: ribonucleic acid hybridization; PTX: pentoxifylline; vit E: vitamin E. day). 80 The beneficial effect of IFN-␥ on skin lesions also was reported in a randomized study: Mouth aperture and skin scores increased significantly in the treated group after 12 months of treatment with IFN-␥ (100 mcg per day).…”