Diabetes-Related Ankyrin Repeat Protein (DARP/Ankrd23) Modifies Glucose Homeostasis by Modulating AMPK Activity in Skeletal Muscle.

Skeletal muscle is the major site for glucose disposal, the impairment of which closely associates with the glucose intolerance in diabetic patients. Diabetes-related ankyrin repeat protein (DARP/Ankrd23) is a member of muscle ankyrin repeat proteins, whose expression is enhanced in the skeletal muscle under diabetic conditions; however, its role in energy metabolism remains poorly understood. Here we report a novel role of DARP in the regulation of glucose homeostasis through modulating AMP-activated protein kinase (AMPK) activity. DARP is highly preferentially expressed in skeletal muscle, and its expression was substantially upregulated during myotube differentiation of C2C12 myoblasts. Interestingly, DARP-/- mice demonstrated better glucose tolerance despite similar body weight, while their insulin sensitivity did not differ from that in wildtype mice. We found that phosphorylation of AMPK, which mediates insulin-independent glucose uptake, in skeletal muscle was significantly enhanced in DARP-/- mice compared to that in wildtype mice. Gene silencing of DARP in C2C12 myotubes enhanced AMPK phosphorylation, whereas overexpression of DARP in C2C12 myoblasts reduced it. Moreover, DARP-silencing increased glucose uptake and oxidation in myotubes, which was abrogated by the treatment with AICAR, an AMPK activator. Of note, improved glucose tolerance in DARP-/- mice was abolished when mice were treated with AICAR. Mechanistically, gene silencing of DARP enhanced protein expression of LKB1 that is a major upstream kinase for AMPK in myotubes in vitro and the skeletal muscle in vivo. Together with the altered expression under diabetic conditions, our data strongly suggest that DARP plays an important role in the regulation of glucose homeostasis under physiological and pathological conditions, and thus DARP is a new therapeutic target for the treatment of diabetes mellitus.

Loss of apoptosis regulator through modulating IAP expression (ARIA) protects blood vessels from atherosclerosis.

Atherosclerosis is the primary cause for cardiovascular disease. Here we identified a novel mechanism underlying atherosclerosis, which is provided by ARIA (apoptosis regulator through modulating IAP expression), the transmembrane protein that we recently identified. ARIA is expressed in macrophages present in human atherosclerotic plaque as well as in mouse peritoneal macrophages. When challenged with acetylated LDL, peritoneal macrophages isolated from ARIA-deficient mice showed substantially reduced foam cell formation, whereas the uptake did not differ from that in wild-type macrophages. Mechanistically, loss of ARIA enhanced PI3K/Akt signaling and consequently reduced the expression of acyl coenzyme A:cholesterol acyltransferase-1 (ACAT-1), an enzyme that esterifies cholesterol and promotes its storage, in macrophages. Inhibition of PI3K abolished the reduction in ACAT-1 expression and foam cell formation in ARIA-deficient macrophages. In contrast, overexpression of ARIA reduced Akt activity and enhanced foam cell formation in RAW264.7 macrophages, which was abrogated by treatment with ACAT inhibitor. Of note, genetic deletion of ARIA significantly reduced the atherosclerosis in ApoE-deficient mice. Oil red-O-positive lipid-rich lesion was reduced, which was accompanied by an increase of collagen fiber and decrease of necrotic core lesion in atherosclerotic plaque in ARIA/ApoE double-deficient mice. Analysis of bone marrow chimeric mice revealed that loss of ARIA in bone marrow cells was sufficient to reduce the atherosclerogenesis in ApoE-deficient mice. Together, we identified a unique role of ARIA in the pathogenesis of atherosclerosis at least partly by modulating macrophage foam cell formation. Our results indicate that ARIA could serve as a novel pharmacotherapeutic target for the treatment of atherosclerotic diseases.

Manipulation of cardiac phosphatidylinositol 3-kinase (PI3K)/Akt signaling by apoptosis regulator through modulating IAP expression (ARIA) regulates cardiomyocyte death during doxorubicin-induced cardiomyopathy.

PI3K/Akt signaling plays an important role in the regulation of cardiomyocyte death machinery, which can cause stress-induced cardiac dysfunction. Here, we report that apoptosis regulator through modulating IAP expression (ARIA), a recently identified transmembrane protein, regulates the cardiac PI3K/Akt signaling and thus modifies the progression of doxorubicin (DOX)-induced cardiomyopathy. ARIA is highly expressed in the mouse heart relative to other tissues, and it is also expressed in isolated rat cardiomyocytes. The stable expression of ARIA in H9c2 cardiac muscle cells increased the levels of membrane-associated PTEN and subsequently reduced the PI3K/Akt signaling and the downstream phosphorylation of Bad, a proapoptotic BH3-only protein. When challenged with DOX, ARIA-expressing H9c2 cells exhibited enhanced apoptosis, which was reversed by the siRNA-mediated silencing of Bad. ARIA-deficient mice exhibited normal heart morphology and function. However, DOX-induced cardiac dysfunction was significantly ameliorated in conjunction with reduced cardiomyocyte death and cardiac fibrosis in ARIA-deficient mice. Phosphorylation of Akt and Bad was substantially enhanced in the heart of ARIA-deficient mice even after treatment with DOX. Moreover, repressing the PI3K by cardiomyocyte-specific expression of dominant-negative PI3K (p110α) abolished the cardioprotective effects of ARIA deletion. Notably, targeted activation of ARIA in cardiomyocytes but not in endothelial cells reduced the cardiac PI3K/Akt signaling and exacerbated the DOX-induced cardiac dysfunction. These studies, therefore, revealed a previously undescribed mode of manipulating cardiac PI3K/Akt signaling by ARIA, thus identifying ARIA as an attractive new target for the prevention of stress-induced myocardial dysfunction.

Ecscr regulates insulin sensitivity and predisposition to obesity by modulating endothelial cell functions.

Insulin resistance is closely associated with obesity and is one of the earliest symptoms of type-2 diabetes. Endothelial cells are involved in the pathogenesis of insulin resistance through their role in insulin delivery and adipose tissue angiogenesis. Here we show that Ecscr (endothelial cell surface expressed chemotaxis and apoptosis regulator; also known as ARIA), the transmembrane protein that regulates endothelial cell signalling, is highly expressed in white and brown adipose tissues, and regulates energy metabolism and glucose homeostasis by modulating endothelial cell functions. Ecscr-deficient mice fed a normal chow show improved glucose tolerance and enhanced insulin sensitivity. We demonstrate that Ecscr deletion enhances the insulin-mediated Akt/endothelial nitric oxide synthase activation in endothelial cells, which increases insulin delivery into the skeletal muscle. Ecscr deletion also protects mice on a high-fat diet from obesity and obesity-related metabolic disorders by enhancing adipose tissue angiogenesis. Conversely, targeted activation of Ecscr in endothelial cells impairs glucose tolerance and predisposes mice to diet-induced obesity. Our results suggest that the inactivation of Ecscr enhances insulin sensitivity and may represent a new therapeutic strategy for treating metabolic syndrome.

Macrophages play a unique role in the plaque calcification by enhancing the osteogenic signals exerted by vascular smooth muscle cells.

Vascular calcification is a major risk factor for the cardiovascular disease, yet its underlying molecular mechanisms remain to be elucidated. Recently, we identified that osteogenic signals via bone morphogenetic protein (BMP)-2 exerted by vascular smooth muscle cells (VSMCs) play a crucial role in the formation of atherosclerotic plaque calcification. Here we report a synergistic interaction between macrophages and VSMCs with respect to plaque calcification. Treatment with conditioned medium (CM) of macrophages dramatically enhanced BMP-2 expression in VSMCs, while it substantially reduced the expression of matrix Gla-protein (MGP) that inhibits the BMP-2 osteogenic signaling. As a result, macrophages significantly accelerated the osteoblastic differentiation of C2C12 cells induced by VSMC-CM. In contrast, macrophage-CM did not enhance the osteoblastic gene expressions in VSMCs, indicating that macrophages unlikely induced the osteoblastic trans-differentiation of VSMCs. We then examined the effect of recombinant TNF-α and IL-1ß on the VSMC-derived osteogenic signals. Similar to the macrophage-CM, both cytokines enhanced BMP-2 expression and reduced MGP expression in VSMCs. Nevertheless, only the neutralization of TNF-α but not IL-1ß attenuated the effect of macrophage-CM on the expression of these genes in VSMCs, due to the very low concentration of IL-1ß in the macrophage-CM. On the other hand, VSMCs significantly enhanced IL-1ß expression in macrophages, which might in turn accelerate the VSMC-mediated osteogenic signals. Together, we identified a unique role of macrophages in the formation of plaque calcification in coordination with VSMCs. This interaction between macrophages and VSMCs is a potential therapeutic target to treat and prevent the atherosclerotic plaque calcification.

Expression levels of thymidylate synthase, dihydropyrimidine dehydrogenase, and thymidine phosphorylase in patients with colorectal cancer.

BACKGROUND: Predictors of the response of colorectal cancer to chemotherapy remain poorly understood. We analyzed the mRNA expression levels of enzymes related to sensitivity to 5-fluorouracil derivatives in patients with colorectal cancer. PATIENTS AND METHODS: Danenberg tumor profile method (DTP) was used in order to measure mRNA expression levels of thymidylate synthase (TYMS), dihydropyrimidine dehydrogenase (DPYD), and thymidine phosphorylase (TYMP) from 180 patients with colorectal cancer. The relations of expression levels with clinicopathological factors and outcomes were studied. RESULTS: Higher TYMS expression was associated with greater age, DPYD expression with greater age, poorer differentiation and low invasion, and TYMP expression with poorer differentiation and lack of peritoneal metastasis. DPYD expression positively correlated with TYMP expression. In patients with stage IV disease, high DPYD or TYMP expression was associated with poor outcomes. CONCLUSION: mRNA expression of TYMS, DPYD, and TYMP is associated with distinct characteristics and may be useful for predicting survival in patients with stage IV colorectal cancer.

Methylation of the DFNA5 gene is frequently detected in colorectal cancer.

BACKGROUND: Recently, the human deafness, autosomal dominant 5 gene, DFNA5, has frequently been detected in cancer tissues. The methylation status of the DFNA5 gene in colorectal cancer was examined and was compared to the clinocopathological findings. MATERIALS AND METHODS: Eighty-five tumor samples and corresponding normal tissues were obtained from patients with colorectal cancer who underwent surgery at our hospital. The methylation status of the DFNA5 gene in these samples was examined by quantitative methylation-specific PCR (qMSP). Subsequently, the clinicopathological findings were correlated with the methylation status of the DFNA5 gene. RESULTS: DFNA5 gene methylation was found in 29 (34%) out of the 85 colorectal carcinomas, suggesting that it was frequently observed in colorectal cancer. A significant correlation with methylation was observed for lymphatic vessel invasion and TNM stage (p=0.0268 and p=0.0189, respectively). CONCLUSION: DFNA5 might act as a tumor suppressor gene and DFNA5 gene methylation might play an important role in the development of colorectal cancer. Our data implicate DFNA5 gene methylation as a novel molecular biomarker in colorectal cancer.

Detection of vimentin methylation in the serum of patients with gastric cancer.

AIM: Detection of gastric cancer using serum assay of vimentin methylation. METHODS: A quantitative methylation-specific polymerase chain reaction assay was used to detect vimentin gene (VIM) methylation in the serum of 71 patients with gastric cancer. RESULTS: Mean VIM methylation in cancer patients (0.304 ± 0.558) was significantly higher than that in healthy donors (0.011 ± 0.015, p=0.018). The sensitivity of VIM methylation (33.8%) was similar to the one of carbohydrate antigen 19-9 (CA19-9) (25.4%), higher than the one of carcinoembryonic antigen (CEA) (12.7%), and significantly higher than the sensitivity of both markers for patients with stage I and IV disease (p=0.010 and 0.044, respectively). At all stages, the sensitivity of a combination of markers was higher than the sensitivity of any in isolation marker and was similar for stages I, II and III, reaching 76.9% for stage IV disease. CONCLUSION: VIM methylation may represent a useful marker for the detection of tumor DNA in the serum of patients with gastric cancer.

Methylation of the homeobox gene, HOPX, is frequently detected in poorly differentiated colorectal cancer.

BACKGROUND: Homeodomein only protein x (HOPX) gene methylation has frequently been detected in cancer tissues. The methylation status of the HOPX gene in colorectal cancer was examined and compared to the clinocopathological findings. MATERIALS AND METHODS: Eighty-nine tumor samples and corresponding normal tissues were obtained from colorectal cancer patients who underwent surgery at our hospital. The methylation status of the HOPX gene in these samples was examined by quantitative methylation-specific PCR (qMSP). Subsequently, the clinicopathological findings were correlated with the methylation status of the HOPX gene. RESULTS: HOPX gene methylation was found in 46 (52%) out of the 89 colorectal carcinomas, suggesting that it was frequently observed in colorectal cancer. A significant increase of methylation was observed in the poorly differentiated carcinomas (p=0.0049). CONCLUSION: HOPX gene methylation could play an important role for the development of colorectal cancer and is closely related to the histological type.

Loss of bcl-2 during the senescence exacerbates the impaired angiogenic functions in endothelial cells by deteriorating the mitochondrial redox state.

Ageing is an important risk factor for ischemic cardiovascular diseases, although its underlying molecular mechanisms remain to be elucidated. Here, we report a crucial role of Bcl-2 in the impaired angiogenic functions in senescent endothelial cells (ECs) by modulating the mitochondrial redox state. Cellular senescence impaired angiogenic functions in ECs without attenuating the mitogen-activated protein kinase or Akt signaling, and vascular endothelial growth factor receptor 2 or Tie-2 expressions. We identified that Bcl-2 expression was markedly reduced in 3 independent models for senescent ECs, and pharmacological inhibition, as well as small interfering RNA-mediated gene silencing of Bcl-2, significantly impaired the angiogenic functions in young ECs. Bcl-2 has an antioxidative role by locating the glutathione at mitochondria, and we found that mitochondrial oxidative stress was significantly augmented in senescent ECs, in association with reduced mitochondria-associated glutathione. Transfection of Bcl-2 in senescent ECs significantly reduced the mitochondrial oxidative stress, restored the mitochondrial membrane potential, and improved the angiogenic capacity. Furthermore, gene transfer of Bcl-2 using adenovirus significantly improved the in vivo angiogenesis in the Matrigel plugs implanted into aged mice, whereas the Bcl-2 inhibitor reduced the angiogenesis in the Matrigel plugs implanted into young mice. Together, Bcl-2 plays a crucial role in the regulation of the mitochondrial redox state in ECs, and, thus, loss of Bcl-2 during the senescence exacerbates the impaired angiogenesis by augmenting the mitochondrial oxidative stress.

[Complete response to S-1/CDDP combination in a patient with obstructive jaundice secondary to lymph node metastasis by gastric cancer - a case report].

A 55-year-old woman was found to have a type-4 lesion centered on the greater curvature of the lower portion of her stomach during an upper gastrointestinal endoscopic examination.A diagnosis of inoperable advanced gastric carcinoma [type 4, tub 2/por, T3 (SE), N3, H0, P1, cStage IV], complicated by pyloric stenosis, liver dysfunction, and obstructive jaundice untreatable by bile drainage, was made.After obtaining the informed consent of the patient and her family and explain- ing that under the circumstances surgery was not indicated, chemotherapy [S-1 (granules) 80 mg/m2, CDDP 60 mg/m2] was selected. After starting treatment, an improvement in liver dysfunction and jaundice was observed, and at the start of the second course, the patient had become capable of oral feeding.The patient was discharged after completion of the second course. No choices associated with evidence exist for treatment of patients with inoperable advanced gastric cancer (complicated by obstructive jaundice), who are not elderly and have good performance status (PS).We report this case in which improvement of activity of daily living (ADL) was achieved relatively safely by treatment with S-1/CDDP, together with a brief discussion based on the literature.

Apoptosis regulator through modulating IAP expression (ARIA) controls the PI3K/Akt pathway in endothelial and endothelial progenitor cells.

Endothelial and endothelial progenitor cells (ECs and EPCs) play a fundamental role in angiogenesis that is essential for numerous physiological and pathological processes. The phosphatase and tensin homolog (PTEN)/ phosphoinositide 3-kinase (PI3K) pathway has been implicated in angiogenesis, but the mechanism in the regulation of this pathway in ECs and EPCs is poorly understood. Here we show that ARIA (apoptosis regulator through modulating IAP expression), a transmembrane protein that we recently identified, regulates the PTEN/PI3K pathway in ECs and EPCs and controls developmental and postnatal angiogenesis in vivo. We found that ARIA is abundantly expressed in EPCs and regulates their angiogenic functions by modulating PI3K/Akt/endothelial nitric oxide synthase (eNOS) signaling. Genetic deletion of ARIA caused nonfatal bleeding during embryogenesis, in association with increased small vessel density and altered expression of various vascular growth factors including angiopoietins and VEGF receptors. Postnatal neovascularization induced by critical limb ischemia was substantially enhanced in ARIA-null mice, in conjunction with more bone marrow (BM)-derived ECs detected in ischemic muscles. Administration of PI3K or NO synthase inhibitor completely abolished the enhanced neovascularization in ARIA(-/-) mice. Mechanistically, we identified that ARIA interacts with PTEN at the intracellular domain independently of the PTEN phosphorylation in its C-terminal tail. Overexpressed ARIA increased PTEN in the membrane fraction, whereas ARIA-silencing reduced the membrane-associated PTEN, resulting in modified PI3K/Akt signaling. Taken together, our findings establish a previously undescribed mode of regulation of the PTEN/PI3K/Akt pathway by ARIA, and reveal a unique mechanism in the control of angiogenesis. These functions of ARIA might offer a unique therapeutic potential.

MACC 1 as a marker for peritoneal-disseminated gastric carcinoma.

BACKGROUND: Recently, metastasis associated with the colon cancer 1 (MACC1) gene was identified by genome-wide search for differentially expressed genes in human colon cancer tissues and metastases. Previously, the MACC1 expression levels were examined in colorectal carcinomas and it was found that MACC1 expression showed significant correlation with peritoneal dissemination and higher stage of TNM classification. MATERIALS AND METHODS: In this study, MACC1 expression levels were analysed in 41 gastric cancer samples using quantitative real-time polymerase chain reaction (QRT-PCR). Results. Distribution of MACC1 expression scores in primary gastric carcinomas was between 0.01 and 4.36 (average ± SD was 1.34 ± 1.31). Subsequently, clinicopathological data were correlated with the MACC1 expression. It was found that MACC1 expression showed significant correlation with peritoneal dissemination (p=0.038). CONCLUSION: These results suggest that MACC1 is more frequently expressed in peritoneal-disseminated gastric carcinomas and may serve as a new parameter for the prognostic prediction of gastric cancer.

MACC1 as a marker for advanced colorectal carcinoma.

BACKGROUND: Recently, Stein et al. identified the metastasis-associated in colon cancer 1 (MACC1) gene by genome-wide search for differentially expressed genes in human colon cancer tissues and metastases. PATIENTS AND METHODS: We analyzed MACC1 expression levels in 52 colorectal cancer samples using quantitative real-time polymerase chain reaction (QRT-PCR). RESULTS: We found that MACC1 expression showed significant correlation with peritoneal dissemination (p=0.042) and higher stage of TNM classification (p=0.007). CONCLUSION: These results suggest that MACC1 is more frequently expressed in advanced colorectal carcinomas.

Left ventricular free wall rupture associated with a combination of acute myocardial infarction and stress-provoked cardiomyopathy: An autopsy case.

A 74-year-old female was admitted to our hospital due to prolonged chest pain that had lasted about 2 h. An electrocardiogram revealed ST-elevation in leads I, aVL, and V3-6, with an increase in myocardial necrosis markers. Emergency coronary angiography was performed, and left ventriculography showed the typical features of apical ballooning, and so a diagnosis of Takotsubo cardiomyopathy (TC) was made. On the 10th day after admission, the patient suddenly went into cardiopulmonary arrest because of a blow-out type left ventricular (LV) free wall rupture. Despite extensive cardiopulmonary resuscitation, the patient died. The autopsy revealed hemopericardium and a perforating wound located in the anterior wall of the LV. It was revealed that the diagonal branch of the coronary artery was occluded, and so a diagnosis of TC coexisting with acute myocardial infarction (AMI) was made. No previous case of TC accompanied by AMI has been reported. We present its clinical course during hospitalization and the result of a histopathologic examination.

PGP9.5 was less frequently methylated in advanced gastric carcinoma.

BACKGROUND/AIMS: Recently, it was examined the methylation status of the Protein Gene Product 9.5 (PGP9.5) gene in primary tumors derived from 49 patients with colorectal cancer and evaluated the correlation between the methylation status and the clinicopathological findings. A significant difference was observed in lymph node metastasis (p = 0.029), suggesting that PGP9.5 was less frequently methylated in metastatic colorectal cancer. This result prompted us to examine the methylation status of the PGP9.5 gene in gastric cancers. METHODOLOGY: It was examined the methylation status of the PGP9.5 gene in primary tumors derived from 30 patients with gastric cancer using qMSP and evaluated the correlation between the methylation status and the clinicopathological findings. RESULTS: Aberrant methylation of the PGP9.5 gene was detected in 5 of 30 (17%) primary gastric cancers. The present results suggested that the aberrant methylation of the PGP9.5 gene was frequently observed in gastric cancers. Subsequently, clinicopathological data were correlated with the methylation results. A significant difference was observed in extent of tumor, lymph node metastasis, and TNM stage (p = 0.034, 0.015, and 0.028, respectively). CONCLUSION: PGP9.5 was less frequently methylated in advanced gastric cancer compared to earlier one.

Subcortical intracerebral hemorrhage caused by mucormycosis in a patient with a history of bone-marrow transplantation.

We report on a rare case of a patient with rhinocerebral mucormycosis that presented as intracerebral hemorrhage (ICH). A 54-year-old man who was immunosuppressed had ophthalmoplegia. Four days later, ICH developed in his left frontal lobe. The ICH was surgically removed totally. Pathology specimen surgically obtained from brain surface adjacent to hematoma cavity showed blood vessels filled with Mucor mycelium. Combined with surgical findings, venous occlusion by Mucor mycelium might be the cause of ICH in the patient.

WNT5A gene is aberrantly methylated from the early stages of colorectal cancers.

BACKGROUND/AIMS: Recently, it has been reported that WNT5A methylation was frequently detected in colorectal cancers. However, the relationship between the WNT5A methylation and the characteristics of these cancers remains unknown. METHODOLOGY: Methylation status of the WNT5A gene was examined in primary carcinomas and the corresponding normal tissues derived from 49 patients with colorectal cancer using quantitative methylation-specific PCR (qMSP) and the correlation between the methylation status and the clinicopathological findings was evaluated. RESULTS: Aberrant methylation of the WNT5A gene was detected in 17 out of the 49 (35%) primary colorectal cancers. The clinicopathological data were correlated with the methylation results. All stages of colorectal cancers presented WNT5A methylation. CONCLUSIONS: WNT5A gene has been methylated from the early stages of colorectal cancers.

MGMT gene is aberrantly methylated from the early stages of colorectal cancers.

BACKGROUND/AIMS: Recently, Herfarth et al. reported that a subset of colorectal cancers was characterized by a specific methylation pattern in the MGMT promoter associated with reduced MGMT expression. METHODOLOGY: Methylation status of the MGMT gene was examined in primary carcinomas and the corresponding normal tissues derived from 48 patients with colorectal cancer using quantitative methylation-specific PCR (qMSP) and the correlation between the methylation status and the clinicopathological findings was evaluated. RESULTS: Aberrant methylation of the MGMT gene was detected in 10 out of the 48 (21%) primary colorectal cancers. The present study results suggested that the aberrant methylation of the MGMT gene was specific and frequently observed in colorectal cancers. The clinicopathological data were correlated with the methylation results. No significant correlations were observed between the presentation of abnormal methylation in the colorectal carcinomas and patient gender or age, maximal tumor size, tumor extent, tumor site, histology, lymph node metastasis, and Dukes stage. CONCLUSION: All stages of colorectal cancers presented MGMT methylation, indicating that the MGMT gene has been methylated from the early stages of colorectal cancers.