Shigetsugu Ohgi

Inhibition of Experimental Abdominal Aortic Aneurysm in the Rat by Use of Decoy Oligodeoxynucleotides Suppressing Activity of Nuclear Factor κB and ets Transcription Factors

Background—Two phenomena, inflammation and matrix degradation, contribute to the progression of abdominal aortic aneurysm (AAA). Importantly, the inflammation is regulated by the transcription factor nuclear factor (NF)–&kgr;B, whereas the destruction and degradation of elastin fibers by matrix metalloproteinases (MMP) are regulated by ets. Thus, we developed a novel strategy to treat AAA by simultaneous inhibition of both NF-&kgr;B and ets by using chimeric decoy oligodeoxynucleotides (ODN). Methods and Results—AAA was induced in rats by transient aortic perfusion with elastase, whereas transfection of decoy ODN was performed by wrapping a delivery sheet containing decoy ODN around the aorta. Gel-mobility shift assay at 7 days after treatment demonstrated that both NF-&kgr;B and ets binding activity were simultaneously inhibited by chimeric decoy ODN. Transfection of chimeric decoy ODN resulted in significant inhibition of the progression of AAA such as aneurysmal dilation at 4 weeks after treatment as compared with control, accompanied by a reduction of MMP expression. Moreover, the destruction of elastin fibers was inhibited in the aorta transfected with chimeric decoy ODN. Importantly, transfection of chimeric decoy ODN demonstrated potent inhibition of aneurysmal dilatation compared with NF-&kgr;B decoy ODN alone, whereas scrambled decoy ODN had no effects. Interestingly, the migration of macrophages was significantly inhibited by chimeric decoy ODN. Conclusions—We demonstrated that inhibition of the progression of AAA was achieved by a novel strategy with chimeric decoy ODN used against NF-&kgr;B and ets in rat model. NF-&kgr;B and ets are considered to play an important role in the pathogenesis of AAA.

Protein expression of matrix metalloproteinases 2 and 9 and tissue inhibitors of metalloproteinase 1 and 2 in papillary thyroid carcinomas.

Abstract. Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) play an important role in tumor invasion and metastasis. There have been only a few studies on the protein expression of MMPs and TIMPs in thyroid carcinomas. Therefore, we investigated the protein expression of MMP-2, MMP-9, TIMP-1 and TIMP-2 in 86 papillary thyroid carcinomas using immunohistochemistry, semiquantitative scoring morphometry of immunohistochemistry, gelatin zymography, and western blotting. We also examined the correlations between the immunohistochemical scores and several clinicopathological parameters. The immunoreactivities of MMP-2, MMP-9, TIMP-1, and TIMP-2 were largely located in the tumor cells or non-tumor follicular cells and to a much lesser extent in the fibroblasts and endothelial cells in the tumor and non-tumor regions. Compared with non-tumor regions, these four proteins tended to be overexpressed in the tumor cells; the overexpression was found in 64 of 86 (74%), 80 of 86 (93%), 79 of 86 (92%), and 64 of 86 (74%) cases for MMP-2, MMP-9, TIMP-1, and TIMP-2, respectively. Gelatin zymography showed distinct bands of MMP-2 and MMP-9 in tumor extracts but vague bands in non-tumor extracts. Western blotting revealed the specific bands of MMP-2 and MMP-9 in both tumor and non-tumor extracts. Morphometric scoring revealed that high expression of these proteins significantly correlated with large tumor size, presence of lymph node metastasis, high clinical stage, high intrathyroidal invasion, and high vascular invasion. These data suggest that MMP-2, MMP-9, TIMP-1, and TIMP-2 proteins and activities are increased in tumors cells of papillary thyroid carcinomas and that they play an important role in the invasion and metastasis of papillary thyroid carcinomas.

Inhibition of development of experimental aortic abdominal aneurysm in rat model by atorvastatin through inhibition of macrophage migration

Recently, atherosclerosis has been considered to be the result of inflammation. Interestingly, hydroxymethylglutaryl-coenzyme (HMG-Co) A inhibitors (statins), which are clinically used as lipid-lowering agents, have been reported to have various anti-inflammatory effects. As abdominal aortic aneurysm (AAA) is a common degenerative condition associated with atherosclerosis, this study was designed to investigate the inhibitory effect of a statin, atorvastatin, on aneurysm formation apart from its lipid-lowering effect. We employed an elastase-induced rat AAA model, as statins do not lower cholesterol in rats. Mean aneurysm diameter was significantly smaller in the atorvastatin treatment group as compared to control at 4 weeks after surgery (P<0.05). Interestingly, atorvastatin inhibited the expression of ICAM and MCP-1, followed by the suppression of macrophage recruitment into the aortic wall at 1 week after operation. A significant reduction in MMP-12, but not MMP-2, -3 and -9, expression was also observed by treatment with atorvastatin at 1 week after surgery. In addition, synthesis of collagen and elastin in the vascular wall were significantly increased by atorvastatin. Here, the present study demonstrated a direct effect of atorvastatin to inhibit the progression of aortic aneurysm, independent of its lipid-lowering effect. This study suggests new therapeutic aspects of statins to inhibit the progression of aneurysms.

Pulmonary embolism in patients with isolated soleal vein thrombosis

This study investigated the features of calf deep vein thrombosis (DVT) as a pulmonary embolic source. Fifty-eight lower limbs in 29 patients who were suspected of having DVT distal to the popliteal vein were screened by ultrasonography. Then, ascending venog raphy was performed to confirm the diagnosis. Pulmonary embolism (PE) was diagnosed in suspected patients by use of pulmonary perfusion scanning or pulmonary angiography. Venography revealed calf DVT in 33 limbs in 28 patients. Of 28 patients, six had symp tomatic PE. Thrombosis was found in the muscle veins in 18 limbs, the trunk veins in 11, and both veins in four. Isolated single vein thrombosis was found in the soleal vein in 14 limbs (42%), the posterior tibial vein in eight, the peroneal vein in two, and the gastroc nemius vein in two. The overall percentage of soleal vein thrombi was 61%. All six patients with symptomatic PE had isolated soleal vein thromboses. Calf DVT was a pulmonary embolic source when isolated thrombosis of the large soleal vein was more than 7 mm in diameter. Soleal veins were the most frequent and important location of calf DVT, suggesting that these were an occasional embolic source of critical PE.

Identification of secernin 1 as a novel immunotherapy target for gastric cancer using the expression profiles of cDNA microarray

Despite the discovery of multiple TAAs, only a limited number is available for clinical application, particularly against epithelial malignancies. In this study we searched for novel TAAs using expression profiles of gastric cancer examined with cDNA microarray, and identified the SCRN1 gene as a candidate. SCRN1 was confirmed to be expressed in five out of seven gastric cancers with semiquantitative RT‐PCR. With Northern blot analysis, it was detected abundantly in the testis and ovary, but it was barely detectable in 14 other normal human adult organs. Colony formation assay revealed that its augmented expression is associated with promoted cell growth. As these expression profiles and functional features of SCRN1 appeared to be compatible with the characteristics of the hypothesized ideal TAAs, we examined whether SCRN1 protein contains antigenic epitope peptides restricted to HLA‐A*0201. We synthesized the candidate peptides derived from SCRN1, and tried to induce CTLs with each peptide. The CTL clones were successfully induced with a peptide SCRN1‐196 (KMDAEHPEL), and they lyzed not only the peptide‐pulsed targets but also the tumor cells expressing both SCRN1 and HLA‐A*0201 endogenously. These results strongly suggest that SCRN1‐196 is an epitope peptide restricted to HLA‐A*0201. Furthermore, we synthesized an anchor‐modified peptide SCRN1–9 V (KMDAEHPEV), in which leucine at position 9 was substituted for valine to increase the binding affinity to the HLA‐A*0201 molecules. The CTL clones induced by SCRN1–9 V also recognized tumor cells expressing its natural SCRN1 protein endogenously. These results strongly suggest that SCRN1 is a novel TAA and these peptides, both native and modified, may be applicable for cancer vaccines to treat gastric cancer. (Cancer Sci 2006; 97: 411–419)

Identification of immunoglobulin superfamily 11 (IGSF11) as a novel target for cancer immunotherapy of gastrointestinal and hepatocellular carcinomas

We previously performed gene expression profile analyses of 20 intestinal‐type gastric cancers, and identified a set of genes whose expression levels were elevated in cancer tissues compared to their corresponding non‐cancerous tissues. In the present study we focused on the immunoglobulin superfamily 11 gene (IGSF11). Its expression was also elevated in colorectal cancers and hepatocellular carcinomas as well as intestinal‐type gastric cancers. Northern blot analysis showed that it was expressed abundantly in testis and ovary. These data suggest that IGSF11 is a good candidate of cancer‐testis antigen. Furthermore, suppression of IGSF11 by siRNA retarded the growth of gastric cancer cells. To investigate the possibility of clinical application of peptide vaccine to IGSF11, we synthesized candidate epitope peptides for IGSF11 and tested whether the peptides elicit IGSF11‐specific CTL. As a result, we successfully established oligo‐clonal CTL by stimulation with IGSF11‐9‐207 (ALSSGLYQC). In addition, we also established additional CTL using IGSF11‐9V (ALSSGLYQV), anchor‐modified peptides of IGSF11‐9‐207. These peptides showed IGSF11‐specific cytotoxic activity in an HLA‐A*0201‐restricted fashion, suggesting that these peptides may be applicable for cancer immunotherapy. These findings have provided a novel insight into carcinogenesis of the stomach, colon and liver, and will be helpful for the development of novel therapeutic strategies to a wide range of human cancers. (Cancer Sci 2005; 96: 498 –506)

Mitochondrial DNA deletion associated with the reduction of adenine nucleotides in human atrium and atrial fibrillation

Background Structural changes in the number, size, and shape of mitochondria (mt) have been observed in the atrial muscles of patients with atrial fibrillation (AF) and of animals with rapid atrial pacing, however, it is not known whether the mitochondrial function is impaired in human atrium with AF.

Effect of different frequencies of tensile strain on human dermal fibroblast proliferation and survival

The aim of this study is to compare the effect of a high‐frequency repetitive (HF) stretch or an intermittent (I) stretch on the cell proliferation and survival of human dermal fibroblasts and to determine the activation of any relevant signal pathways. Cultured human dermal fibroblasts were exposed to either HF or I stretch. Cell number was measured by counting, while DNA synthesis was assessed by 5‐bromo‐2′‐deoxyuridine (BrdU) staining and apoptosis by terminal deoxynucleotidyl transferase‐mediated dUTP nick‐end labeling staining. To investigate the potential mechanisms of repetitive strain on the proliferation and survival of fibroblasts, the activation of relevant transduction pathways, such as p38 mitogen‐activated protein kinase (MAPK), extracellular signal‐regulated kinase (ERK)1/2, AKT, and BAD, was assessed by Western blot. In addition, the effect of inhibition of these pathways on the fibroblast response was also studied. After either HF or I stretch for 7 days, fibroblast number was significantly decreased and there were less BrdU‐positive cells. The numbers of apoptotic and/or necrotic fibroblasts were not affected. p38 MAPK and ERK1/2 were significantly activated after HF stretch, but AKT and BAD were significantly activated after I stretch. The inhibitors of p38 MAPK and MAPK/ERK kinase as well as dominant‐negative AKT reduced cell number after both HF and I stretch but these pathways were not critical for the stretch‐induced decrease in cell number.

Inhibition of ets, an essential transcription factor for angiogenesis, to prevent the development of abdominal aortic aneurysm in a rat model

The pathophysiology of abdominal aortic aneurysms (AAA) is considered to be complicated. As matrix degradation contributes to the progression of AAA, the destruction and degradation of elastin fibers caused by an increase in matrix metalloproteinases (MMPs) plays a pivotal role in the development of AAA. Although ets, an essential transcription factor for angiogenesis, regulates MMPs, the role of ets in the development of AAA has not yet been clarified. Thus, we evaluated the role of ets in a rat AAA model using a decoy strategy. Transfection of ODN into AAA was performed by transient aortic perfusion of elastase and by wrapping the AAA in a delivery sheet containing decoy ODN. The inhibitory effect of ets decoy ODN on ets binding activity was confirmed by gel mobility shift assay. MMPs expression was decreased in the aorta transfected with ets decoy ODN as compared to scrambled decoy ODN. Also, ultrasound study demonstrated that elastase-induced aneurismal dilation was significantly suppressed by transfection of ets decoy ODN at 4 weeks after treatment as compared to scrambled decoy ODN. Moreover, the destruction of elastin fibers was inhibited in the aorta transfected with ets decoy ODN, accompanied by a reduction of MMPs expression. An inhibitory effect of decoy ODN on MMP expression was confirmed by ex vivo experiments showing that transfection of decoy ODN into an organ culture of human aorta resulted in significant inhibition of the secretion of both MMP-1 and MMP-9. Here, we demonstrated that ets may play a pivotal role in the progression of AAA through the activation of MMPs in a rat model. Ets might be a potential target to develop pharmacotherapy/gene therapy to treat AAA through the inhibition of MMPs.

Expression of RUNX3 protein in human lung adenocarcinoma: implications for tumor progression and prognosis.

Runt‐related transcription factor 3 belongs to the runt domain family of transcription factors that play a pivotal role during normal tissue development and tumorigenesis in several organs. We directed our attention to the expression of RUNX3 protein in human lung AC and non‐neoplastic lung tissues, comparing the results with clinicopathological profiles. We evaluated the expression of RUNX3 protein in 17 pairs of lung AC and non‐neoplastic lung tissue. Furthermore, 98 lung AC were studied to examine the frequency of RUNX3‐positive cells. Western blot analysis showed a single band at 45 kDa in all 17 AC and non‐neoplastic tissues. Immunohistochemistry revealed immunoreactivity in alveolar type II pneumocytes or Clara cells. RUNX3 was expressed more frequently in the carcinomas with a BAC component than in those without (P < 0.01). Lower RUNX3 levels were associated with poorly differentiated types (P = 0.049). The five‐year survival rate was significantly higher in the 50 patients with higher levels of RUNX3 expression than in the 48 patients with lower levels (P = 0.027). The expression of RUNX3 protein in lung AC might play a pivotal role in tumor progression and patients’ survival. (Cancer Sci 2005; 96: 227–231)