No abstract

In a previously reported attempt to regenerate small intestine with autologous tissues, collagen scaffolds were used without cell seeding or with autologous mesenchymal stem cell seeding. However the regenerated intestine lacked a smooth muscle layer. To accomplish regeneration of a smooth muscle layer, this present study used collagen scaffolds seeded with the smooth muscle cells (SMC) in a canine model. Autologous SMC were isolated from stomach wall and cultured. Two types of scaffolds were fabricated: in SMC (+), cultured SMCs were mixed with collagen solution and poured into a collagen sponge; and in SMC (-), SMCs were omitted. Both scaffolds were implanted into defects of isolated ileum as a patch graft. Animals were euthanized at 4, 8, and 12 weeks; for the last time point, the ileal loop had been reanastomosed at 8 weeks. At 12 weeks, the SMC (-) group showed a luminal surface covered by a regenerated epithelial cell layer with very short villi; however only a thin smooth muscle layer was observed, representing the muscularis mucosae. In the SMC (+) group, the luminal surface was covered completely by a relatively well-developed epithelial layer with numerous villi. Implanted SMCs were seen in the lamina propria and formed a smooth muscle layer. Thus, we concluded that collagen sponge scaffolds seeded with autologous SMCs have a potential for small intestine regeneration.

Purpose: Our previous results suggested that a lackof RUNX3 function contributed tohumangastric carcinogenesis, but the role of RUNX3 inprogression and metastasis remains unclear.We examined RUNX3 expression in clinical samples of peritoneal metastases in gastric cancers. Changes in metastatic potential were assessed in animal experiments using stable RUNX3 transfectants of gastric cancer cells. Finally, global expression changes were analyzed using a cDNA microarray. Experimental Design and Results: Significant down-regulation of RUNX3 through methylation on the promoter region was observed in primary tumors (75%) as well as in all clinical peritoneal metastases of gastric cancers (100 %) compared with normal gastric mucosa. Stable transfection of RUNX3 inhibited cell proliferation slightly, and modest transforming growth factor-h (TGF-h)^induced antiproliferative and apoptotic effects were observed. Interestingly, it strongly inhibited peritoneal metastases of gastric cancers in animal model (P < 0.01). Furthermore, we did globally analyzed expression profiles of f21,000 genes in parent cells and stable transfectant of RUNX3 using a cDNA microarray. Microarray analysis identified f28 candidate genes under the possible downstream control of RUNX3, some of these genes were considered to be possibly involved in peritoneal metastases, which were related to signal transduction (vav3, TOLL-like receptor, MAPKK, MET, S1 00A11, and cathepsin E), apoptosis (caspase 9), immune responses (CD55 and TLR1O), and cell adhesion (sialyltransferase 1 and galectin 4). Some of the genes are involved in theTGF-h signaling pathway. Conclusion: These results indicate that silencing of RUNX3 affects expression of important genes involved in aspects of metastasis including cell adhesion, proliferation, apoptosis, and promoting the peritoneal metastasis of gastric cancer. Identification of such genes could suggest new therapeutic modalities and therapeutic targets.

Radiotherapy is an effective treatment for some esophageal cancers, but the molecular mechanisms of radiosensitivity remain unknown. RUNX3, a novel tumor suppressor of gastric cancer, functions in transforming growth factor (TGF)-b-dependent apoptosis. We obtained paired samples from 62 patients with advanced esophageal cancers diagnosed initially as T3 or T4 with image diagnosis; one sample was obtained from a biopsy before presurgical radiotherapy, and the other was resected in surgical specimens after radiotherapy. RUNX3 was repressed in 67.7% cases of the pretreatment biopsy samples and 96.7% cases of the irradiated, resected samples. The nuclear expression of RUNX3 was associated with radiosensitivity and a better prognosis than cytoplasmic or no RUNX3 expression (Po0.003); cytoplasmic RUNX3 expression was strictly associated with radioresistance. RUNX3 was downregulated and its promoter was hypermethylated in all radioresistant esophageal cancer cell lines examined. Stable transfection of esophageal cancer cells with RUNX3 slightly inhibited cell proliferation in vitro, enhanced the antiproliferative and apoptotic effects of TGF-b and increased radiosensitivity in conjunction with Bim induction. In contrast, transfection of RUNX3-expressing cells with a RUNX3 antisense construct or a Bim-specific small interfering RNA induced radioresistance. Treatment with 5-aza-2 0 -deoxycytidine restored RUNX3 expression, increased radiosensitivity and induced Bim in both control and radioresistant cells. These results suggest that RUNX3 silencing promotes radioresistance in esophageal cancers. Examination of RUNX3 expression in pretreatment specimens may predict radiosensitivity, and induction of RUNX3 expression may increase tumor radiosensitivity.

Objective-Tumor necrosis factor (TNF)-␣-induced endothelial injury, which is associated with atherosclerosis, is mediated by intracellular reactive oxygen species. Iron is essential for the amplification of oxidative stress. We tested whether TNF-␣ accelerated iron accumulation in vascular endothelium, favoring synthesis of hydroxyl radical. Methods and Results-Diverse iron transporters, including iron import proteins (transferrin receptor [TfR] and divalent metal transporter 1 [DMT1]) and an iron export protein (ferroportin 1 [FP1]) coexist in human umbilical endothelial cells (HUVECs). TNF-␣ caused upregulation of TfR and DMT1 and downregulation of FP1, which were demonstrated in mRNA as well as protein levels. These changes in iron transporters were accompanied by accumulation of iron that was both transferrin-dependent and transferrin-independent. Modifications of these mRNAs were regulated posttranscriptionally, and were coordinated with activation of binding activity of iron regulatory protein 1 to the iron responsive element on transporter mRNAs. Using a salicylate trap method, we observed that only simultaneous exposure of endothelial cells to iron and TNF-␣ accelerated hydroxyl radical production. Conclusions-TNF-␣ could cause intracellular iron sequestration, which may participate importantly in the pathophysiology of atherosclerosis and cardiovascular disease. Key Words: cytokines Ⅲ endothelium Ⅲ free radicals Ⅲ inflammation Ⅲ iron A therosclerosis now is generally recognized as a chronic inflammatory condition, and inflammatory cytokines such as tumor necrosis factor (TNF)-␣ have been associated with the development of atherosclerotic lesions and consequent cardiovascular events. 1,2 Dysfunction and loss of vascular endothelial cells, which provide a nonthrombogenic surface and a permeability barrier, occur early in atherosclerosis. 3 Several lines of evidence has suggested that TNF-␣-induced cell injury is mediated through its ability to promote intracellular reactive oxygen species (ROS) formation. 4,5 Among these species, superoxide anion (O 2 Ϫ ) and hydrogen peroxide (H 2 O 2 ) are not very reactive, and usually are neutralized by an elaborate antioxidant defense system. However, transition metal-catalyzed Haber-Weiss reaction can transform O 2 Ϫ and H 2 O 2 to hydroxyl radical, which are extremely powerful oxidizing species. 6,7 Iron is an essential element required for biochemical reactions subserving a wide variety of functions in cells and organisms. 8 However, free iron, possibly the most important transition metal in biologic systems, can act as an electron donor. 6,9 Excessive intracellular accumulation of iron therefore could amplify the damaging effect of oxidative stress in inflammatory conditions, leading to cell injury.The recent identification of iron transport proteins has rapidly expanded our knowledge of molecular aspects of iron processing, especially in the reticuloendothelial system and small intestine. 10,11 Such proteins include transferrin receptor (TfR), divalent metal tr...