Ching-Ching Sung

Expression of fibrogenic cytokines in rat small intestine after fractionated irradiation

The molecular and cellular mechanisms that regulate the radiation-induced fibrotic response in the intestine are not known. In addition to increased amounts of connective tissue, inflammatory cell aggregates are often found, especially in conjunction with acute or chronic mucosal ulcerations. These inflammatory cells are a major source of cytokines that influence connective tissue metabolism. Hence, a possible link may exist between the cellular inflammatory response and fibrosis. This preclinical study examined the influence of fractionated irradiation on the expression of three inflammatory/fibrogenic cytokines in rat small intestine. A rat intestinal transposition model was used for localized fractionated irradiation of a 3-4-cm segment of small bowel. Fifty-nine male Sprague-Dawley rats were irradiated or sham irradiated with 9 daily fractions of 5.2 Gy. Expression of Interleukin 1 alpha (IL-1 alpha), Transforming growth factor beta 1 (TGF-beta 1), and Platelet derived growth factor-AA (PDGF-AA) was assessed by immunohistochemistry. Irradiated and unirradiated intestine was examined 24 h, 14 days, and 26 weeks after completion of irradiation. Unirradiated intestine exhibited immunohistochemical expression of IL-1 alpha, TGF-beta 1 and PDGF-AA that conformed to known staining patterns in normal tissue. Irradiated intestine showed increased expression of all three cytokines at all assessment times. The increased cytokine expression correlated with fibrosis and inflammatory cell infiltrates in irradiated intestine. This was particularly evident in areas with mucosal ulcerations. Fractionated irradiation of small intestine elicits increased expression of IL-1 alpha, TGF-beta 1, and PDGF-AA in areas of acute and chronic radiation injury.(ABSTRACT TRUNCATED AT 250 WORDS)

Is the loss of endothelial thrombomodulin involved in the mechanism of chronicity in late radiation enteropathy

BACKGROUND AND PURPOSE Radiation enteropathy is characterized by locally elevated levels of inflammatory and fibrogenic cytokines. Microvascular injury may sustain these alterations through persistent local hypercoagulopathy, platelet aggregation, leukocyte adhesion and release of biologically active mediators. This study assessed the relationship of endothelial thrombomodulin (TM), a key regulator of the protein C anticoagulant pathway and marker of endothelial function, with transforming growth factor beta (TGF-beta) immunoreactivity and morphologic alterations in radiation enteropathy. MATERIALS AND METHODS Small bowel resection specimens from 9 patients with radiation enteropathy were analyzed by computerized quantitative immunohistochemistry using antibodies against TM, von Willebrand factor (vWF) and TGF-beta. Identical measurements were performed on intestinal resection specimens from otherwise healthy penetrating trauma victims and on archived small intestines. A previously validated image analysis technique was used to assess submucosal vessels for TM and vWF immunoreactivity, and the intestinal wall for total extracellular matrix-associated TGF-beta immunoreactivity. RESULTS Specimens from irradiated patients showed prominent submucosal and subserosal thickening and fibrosis, and obliterative vasculopathy. Control specimens were histopathologically normal. Vascular density and vWF immunoreactivity were similar in radiation enteropathy patients and controls. The image-analysis techniques were highly reproducible, with correlation coefficients for repeated measurements ranging from 0.86 to 0.93. Radiation enteropathy specimens exhibited a highly significant reduction in the number and proportion of TM-positive submucosal vessels per unit area (P < 0.0001) and increased intestinal wall TGF-beta immunoreactivity (P = 0.002). CONCLUSIONS These data support the theory that sustained endothelial dysfunction is involved in the molecular pathogenesis of radiation enteropathy, and point to TM as important in the chronic nature of radiation enteropathy and a potential target for prophylactic and therapeutic interventions.

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

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.

Association of transforming growth factor β (TGF-β) immunoreactivity with specific histopathologic lesions in subacute and chronic experimental radiation enteropathy

Irradiated intestine consistently exhibits increased immunoreactivity of transforming growth factor beta-1 (TGF-beta 1). It is not known whether this increase occurs secondary to mucosal barrier disruption (consequential injury) or to injury in late-responding tissue compartments (primary radiation enteropathy). This study therefore assessed the association between TGF-beta immunoreactivity and specific consequential and primary histopathologic alterations. A small bowel loop was fixed inside the scrotum in male rats and subsequently exposed to either 18 daily fractions of 2.8 Gy or nine daily fractions of 5.6 Gy orthovoltage X-radiation. Radiation-induced induced intestinal complications were recorded and groups of animals were euthanized 2 and 26 weeks post-irradiation. Radiation injury was assessed with a histopathologic radiation injury score (RIS). Total TGF-beta was detected immunohistochemically and measured with interactive computerized image analysis. The image analysis technique yielded highly reproducible quantitation data. The 2.8-Gy group maintained mucosal integrity and had fewer intestinal complications, lower RIS and lower TGF-beta levels than the 5.6-Gy group. There was highly significant correlation between TGF-beta immunoreactivity and radiation injury at both observation times (P < 0.001 and P < 0.0001). At 2 weeks, TGF-beta immunoreactivity correlated with mucosal ulceration (P = 0.002), epithelial atypia (P = 0.005), and serosal thickening (P = 0.0004). At 26 weeks, TGF-beta levels correlated significantly with six of seven histopathologic parameters, most strikingly with vascular sclerosis (P = 0.0003). We conclude that mucosal barrier breakdown is closely associated with increased TGF-beta immunoreactivity in consequential radiation enteropathy. The highly significant correlation between TGF-beta expression levels and alterations in late-responding tissue compartments also suggest a role for TGF-beta in primary radiation enteropathy.

Increased transforming growth factor β (TGF-β) immunoreactivity is independently associated with chronic injury in both consequential and primary radiation enteropathy

PURPOSE Radiation enteropathy is characterized by sustained increase in transforming growth factor beta (TGF-beta) immunoreactivity and connective tissue mast cell (CTMC) hyperplasia that may be responsible for progressive fibrosis and lead to clinical complications. We examined to what extent these chronic molecular and cellular phenomena are associated with acute mucosal breakdown (consequential injury) and/or direct (primary) radiation injury in late-responding compartments. METHODS AND MATERIALS Rat small intestine was exposed to 50.4 Gy x-irradiation given either over 18 days (2.8 Gy daily or 5.6 Gy every other day) or 9 days (2.8 Gy twice daily or 5.6 Gy daily). Intestinal complications were recorded and groups of animals were euthanized at 2 and 26 weeks to assess subacute and chronic injury. Histopathologic changes were assessed with a radiation injury scoring system (RIS), total TGF-beta immunoreactivity was quantified with computerized image analysis, and CTMC hyperplasia was assessed in toluidine blue-stained sections. RESULTS TGF-beta immunoreactivity and CTMC hyperplasia colocalized in areas of injury and were highly significantly correlated. Increased fraction size and decreased overall treatment time were associated with increased RIS (p < 0.01 and p < 0.00001), increased TGF-beta immunoreactivity (p = 0.01 andp < 0.001), and degree of CTMC hyperplasia (p = 0.01 and p < 0.001). Postradiation CTMC numbers increased across treatment groups from 2 to 26 weeks (p < 0.01). TGF-beta immunoreactivity was independently associated with chronic intestinal wall fibrosis (p = 0.003). CONCLUSION This in vivo study supports in vitro evidence linking increased TGF-beta immunoreactivity and mast cell hyperplasia and strongly suggests their involvement in the molecular pathogenesis of both primary and consequential radiation enteropathy.

The synthetic somatostatin analogue, octreotide, ameliorates acute and delayed intestinal radiation injury

PURPOSE Reducing intraluminal proteolytic activity attenuates intestinal radiation toxicity. This study assessed whether pharmacological inhibition of exocrine pancreatic secretion protects against early and delayed radiation enteropathy in a preclinical rat model. METHODS AND MATERIALS Rat ileum was sham-irradiated or exposed to 16 once-daily 4.2 Gy fractions of X-radiation. Vehicle or somatostatin analogue (octreotide, 2 microg/kg/hr) were administered from 2 days prior to 10 days after the end of irradiation. Mucosal injury was monitored noninvasively by assessment of granulocyte transmigration. Radiation injury was assessed at 2 weeks (early phase) and 26 weeks (chronic phase) using quantitative histopathology, immunohistochemistry, and morphometry. RESULTS Octreotide decreased granulocyte transmigration (p<0.0006), reduced accumulation of myeloperoxidase-positive cells at 2 weeks (p = 0.0002), attenuated structural injury at 2 weeks (p = 0.04) and 26 weeks (p = 0.02), preserved mucosal surface area at 2 weeks (p = 0.0008) and 26 weeks p = 0.0008), and reduced intestinal wall thickening at 26 weeks (p = 0.002). Octreotide did not affect granulocyte transmigration, histology, or mucosal surface area in sham-irradiated controls. CONCLUSION These results demonstrate the importance of consequential mechanisms in the pathogenesis of chronic radiation enteropathy. Short-term octreotide administration ameliorates acute radiation-induced mucosal injury, as well as chronic structural changes, and should be subject to further preclinical and clinical testing.

Clinical significance of increased gelatinolytic activity in the rectal mucosa during external beam radiation therapy of prostate cancer

PURPOSE Rectal toxicity (proctitis) is a dose-limiting factor in pelvic radiation therapy. Mucosal atrophy, i.e., net extracellular matrix degradation, is a prominent feature of radiation proctitis, but the underlying mechanisms are not known. We prospectively examined changes in matrix metalloproteinase (MMP)-2 and MMP-9 (gelatinase A and B) in the rectal mucosa during radiation therapy of prostate cancer, as well as the relationships of these changes with symptomatic, structural, and cellular evidence of radiation proctitis. METHODS AND MATERIALS Seventeen patients scheduled for external beam radiation therapy for prostate cancer were prospectively enrolled. Symptoms of gastrointestinal toxicity were recorded, and endoscopy with biopsy of the rectal mucosa was performed before radiation therapy, as well as 2 and 6 weeks into the treatment course. Radiation proctitis was assessed by endoscopic scoring, quantitative histology, and quantitative immunohistochemistry. MMP-2 and MMP-9 were localized immunohistochemically, and activities were determined by gelatin zymography. RESULTS Symptoms, endoscopic scores, histologic injury, and mucosal macrophages and neutrophils increased from baseline to 2 weeks. Symptoms increased further from 2 weeks to 6 weeks, whereas endoscopic and cellular evidence of proctitis did not. Compared to pretreatment values, there was increased total gelatinolytic activity of MMP-2 and MMP-9 at 2 weeks (p = 0.02 and p = 0.004, respectively) and 6 weeks (p = 0.006 and p = 0.001, respectively). Active MMP-2 was increased at both time points (p = 0.0001 and p = 0.002). Increased MMP-9 and MMP-2 at 6 weeks was associated with radiation-induced diarrhea (p = 0.007 and p = 0.02, respectively) and with mucosal neutrophil infiltration (rho = 0.62). CONCLUSIONS Pelvic radiation therapy causes increased MMP-2 and MMP-9 activity in the rectal mucosa. These changes correlate with radiation-induced diarrhea and granulocyte infiltration and may contribute to abnormal connective tissue remodeling in radiation proctitis.

Differential effect of radiation on endothelial cell function in rectal cancer and normal rectum

BACKGROUND Chronic radiation injury of the intestine is associated with significant underexpression of a potent physiological anticoagulant, endothelial cell thrombomodulin (TM). This study compared early and late radiation-induced changes in endothelial TM, urokinase plasminogen activator (uPA), and transforming growth factor beta (TGF-beta) in normal rectum and tumors. METHODS Rectal resection specimens from 27 patients were analyzed: Nine patients underwent primary resection of rectal cancer, 11 tumors were resected after neo-adjuvant radiotherapy, and 7 because of local recurrence after prior resection and adjuvant radiotherapy. TM, uPA, and extracellular matrix-associated TGF-beta, immunoreactivity were assessed using computerized image analysis. RESULTS Multivariate analysis revealed that tumors had more TM-positive vessels (P = 0.003), more uPA-positive cells (P <0.001), and higher TGF-beta immunoreactivity levels (P <0.001) than normal rectum. Preoperative irradiation was associated with decreased proportions of TM-positive vessels in tumors (P = 0.003) and normal rectum (P <0.001). Irradiated tumors had fewer uPA-positive cells (P = 0.003) and less TGF-beta immunoreactivity (P = 0.001) than unirradiated tumors. The proportion of TM-positive vessels in irradiated rectum from patients with recurrence was decreased (P = 0.03), whereas the recurrent (ie, unirradiated) tumors did not differ from primary tumors in terms of TM, TGF-beta, or uPA immunoreactivity. CONCLUSIONS The results support a role for endothelial dysfunction in the pathogenesis of radiation proctitis. Maintaining endothelial cell anticoagulant function may be a potential method to optimize the therapeutic ratio of adjuvant radiotherapy of rectal cancer.

Changes in transforming growth factor β1 gene expression and immunoreactivity levels during development of chronic radiation enteropathy

Chronic intestinal radiation injury is associated with locally increased TGF-beta1 immunoreactivity that correlates with morphological alterations. However, the underlying mechanisms are not known. This study examined changes in intestinal TGF-beta1 immunoreactivity, steady-state TGF-beta1 mRNA levels, and cellular localization of TGF-beta1 mRNA during development of chronic radiation enteropathy in a rat model. A loop of small bowel was fixed inside the scrotum of orchiectomized male rats. The intestine was subsequently exposed locally to 0, 12 or 21 Gy X radiation. Intestine was procured at 24 h and 2, 6 and 26 weeks and subjected to histopathological analysis, quantitative immunohistochemistry with computerized image analysis, assessment of steady-state TGF-beta1 mRNA levels with quantitative reverse transcriptase polymerase chain reaction, and identification of cell types expressing TGF-beta1 mRNA with in situ hybridization. Intestine from the 21-Gy group exhibited more histopathological injury and increased TGF-beta immunoreactivity 2-26 weeks after irradiation compared to the 12-Gy group and sham-irradiated controls. TGF-beta1 mRNA in irradiated intestine increased up to six times relative to controls at 24 h and 2 weeks, was less at 6 weeks, and did not differ from controls at 26 weeks. In situ hybridization detected TGF-beta1 mRNA in epithelial and Paneth cells in control intestine. Irradiated intestine exhibited additional TGF-beta1 mRNA in inflammatory and fibroblast-like cells. We conclude that there is a radiation-induced shift in the cellular sources of TGF-beta1, and that Tgfb1 gene expression is increased mainly during the early phases of radiation enteropathy, preceding the increase in immunoreactivity and histopathological injury. Translational or post-translational mechanisms are likely involved in sustaining increased TGF-beta1 immunoreactivity levels during the chronic phase of radiation enteropathy.

Upregulation and spatial shift in the localization of the mannose 6-phosphate/insulin-like growth factor II receptor during radiation enteropathy development in the rat.

BACKGROUND AND PURPOSE Transforming growth factor beta1 (TGF-beta1) appears to play an important role in the pathogenesis of chronic radiation-induced fibrosis in the intestine and several other organs. TGF-beta1 is secreted as a non-biologically active complex and its function depends on activation. In vitro data suggest that the mannose 6-phosphate/insulin-like growth factor-beta (M6P/IGF-II) receptor is involved in the mechanism of TGF-beta1 activation. Thus, we used a rat model of radiation enteropathy to examine the potential role of the M6P/IGF-II receptor in the in vivo regulation of TGF-beta1 activity and localization. MATERIALS AND METHODS A scrotal hernia containing a loop of small intestine was created in male rats. The intestine in the scrotum was exposed to 0, 12, or 21 Gy single dose X-radiation. Groups of rats were euthanized 1 day and 2, 6 and 26 weeks after irradiation. Histopathologic injury was assessed with a radiation injury score (RIS). Computerized image analysis was used to identify M6P/IGF-II receptor-positive cells and to quantify extracellular matrix-associated TGF-beta1 immunoreactivity. Changes in urokinase plasminogen activator (uPA), tissue-like plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1) immunoreactivity were also assessed. RESULTS In normal (sham-irradiated) intestine, M6P/IGF-II immunoreactivity was confined to relatively weak, but specific epithelial staining. Irradiated intestine exhibited a highly significant time- and dose-dependent increase in the number of M6P/IGF-II receptor-positive cells (P < 0.001). There was a striking spatial shift of M6P/IGF-II receptor immunoreactivity from epithelium during the early post-radiation phase to stromal cells, most notably fibroblasts during the later stages of injury. Irradiated intestine exhibited distinct co-localization of M6P/ IGF-II receptor-positive cells and extracellular matrix-associated TGF-beta1 in areas of histopathologic injury. There were highly significant associations between the number of M6P/IGF-II receptor-positive stromal cells and TGF-beta1 immunoreactivity (P < 0.001), radiation-induced fibrosis (P < 0.001) and RIS (P < 0.001). Endothelial tPA immunoreactivity decreased significantly after irradiation (P < 0.001), whereas uPA and PAI-1 immunoreactivity levels appeared to be unchanged. CONCLUSIONS M6P/IGF-II receptor upregulation may be a key factor in the in vivo control of TGF-beta1 activity and responsible for the tissue specificity of TGF-beta1 action after irradiation.