Cytology-based Detection of Circulating Tumour Cells in Human Pancreatic Cancer Xenograft Models With KRAS Mutation.

BACKGROUND/AIM: To examine the dynamics of circulating tumour cells (CTCs) in pancreatic cancer (PC), new mouse CTC models from human PC xenografts were developed. MATERIALS AND METHODS: Orthotopic (pancreas) and heterotopic (subcutaneous) transplantation models using GFP-tagged SUIT-2 PC cells were prepared. Using a cytology-based CTC detection platform, CTCs and metastasis were compared. RESULTS: The two types of orthotopic models, including the surgical transplantation model and the intraperitoneal injection model, showed a similar pattern of initial pancreatic tumour formation and subsequent development of peritoneal and hematogenous lung metastases. In the heterotopic model, only hematogenous lung metastasis was observed, and the number of CTCs and lung metastases was higher than that of the orthotopic model. Furthermore, KRAS mutation (G12D) was detected in CTCs. CONCLUSION: These orthotopic and heterotopic models clearly differ in terms of the pattern of metastasis and CTCs and therefore, would be useful PC models to investigate the effect of drug-therapy on CTCs and the role of KRAS mutation.

Clinical prediction rule for neurological sequelae due to acute encephalopathy: a medical community-based validation study in Harima, Japan.

OBJECTIVES: This study aimed to verify the screening performance of our clinical prediction rule for neurological sequelae due to acute encephalopathy (NSAE-CPR), which previously identified the following three variables as predictive of poor outcomes: (1) refractory status epilepticus; (2) consciousness disturbance and/or hemiplegia at 6 hours from onset and (3) aspartate aminotransferase >90 IU/L within 6 hours of onset. DESIGN: Medical community-based multicentre retrospective cohort study. SETTING: Six regional hospitals in Harima and one tertiary centre in Kobe, Japan, from 2008 to 2012. PARTICIPANTS: We enrolled a total of 1612 patients aged <16 years who met the diagnostic criteria for an initial diagnosis of complex febrile seizure. Patients with a history of neurological disease and those included in the derivation cohort were excluded. PRIMARY OUTCOME MEASURES: Univariate and multivariate analyses were performed to determine the association between each of the three predictor variables and poor AE outcome (Pediatric Cerebral Performance Category score ≥2). Receiver operating characteristic curve (ROC) analysis was also performed to assess the screening performance of the NSAE-CPR. RESULTS: The ROC analysis identified at least one of the three predictive variables as an optimal cut-off point, with an area under the curve of 0.915 (95% CI 0.825 to 1.000). The sensitivity, specificity, positive and negative predictive values, positive and negative likelihood ratios and Matthews correlation coefficient were 0.867, 0.954, 0.149, 0.999, 18.704, 0.140 and 0.349, respectively. CONCLUSIONS: Our findings indicate that the NSAE-CPR can be used for the screening and identification of patients with poor outcomes due to acute encephalopathy within 6 hours of onset.

Suppression of abdominal aortic aneurysm formation by AR-R17779, an agonist for the α7 nicotinic acetylcholine receptor.

OBJECTIVE: Activation of vagal nerve suppresses inflammatory responses through activation of α7 nicotinic acetylcholine receptor (nAchR). We sought to determine whether AR-R17779, a selective agonist of α7nAchR, affects the development of abdominal aortic aneurysm (AAA). METHODS AND RESULTS: AAA was induced by topical application of calcium chloride (CaCl2) to abdominal aorta (AAA group). NaCl (0.9%) was substituted for CaCl2 as a sham operation (SHAM group). AR-R17779 was administered in drinking water (AAA/AR-R group). One and 6 weeks after the operation, aortic tissue was excised for histological and molecular analyses. Aortic diameter and macrophage infiltration into the aortic adventitia were increased in AAA group compared with SHAM group at 6 weeks. Treatment with AR-R17779 reduced the diameter of the aorta and macrophage infiltration compared with AAA group. Wavy morphology of the elastic lamellae was lost in AAA group while it was preserved in AAA/AR-R group. Expression of inflammatory cytokines and matrix metalloproteinase (MMP) activities were enhanced in AAA group, which was suppressed in AAA/AR-R group. AR-R17779 treatment suppressed CaCl2-induced expression of cytokines, activities of MMPs and NF-κB activation at 1 week when aortic dilatation had not developed. CONCLUSION: Treatment with AR-R17779 prevented the enlargement of abdominal aorta induced by CaCl2 in association with reduced inflammation and extracellular matrix disruption. These findings suggest therapeutic potential of α7nAchR activation for prevention of AAA development.

Stimulation of α7 nicotinic acetylcholine receptor by AR-R17779 suppresses atherosclerosis and aortic aneurysm formation in apolipoprotein E-deficient mice.

Atherosclerosis is a chronic inflammatory disease. It has been appreciated that vagus nerve inhibits macrophage activation via α7 nicotinic acetylcholine receptor (nAChR), termed the cholinergic anti-inflammatory pathway. We explored the effects of AR-R17779, a selective α7nAChR agonist, on atherosclerosis and aneurysm formation in apolipoprotein E (ApoE)-deficient mice. ApoE-deficient mice were fed a high-fat diet (HFD) and angiotensin II (Ang II) was infused by osmotic minipumps from 10-week-old for 4weeks. AR-R17779 was given in drinking water ad libitum. Oil red O staining of the aorta showed that combined loading of HFD and Ang II induced marked atherosclerosis compared with control mice fed a normal chow. Treatment with AR-R17779 significantly reduced atherosclerotic plaque area and improved survival of mice. Treatment with AR-R17779 also suppressed abdominal aortic aneurysm formation. Quantitative RT-PCR of the aorta revealed that mRNA expression levels of interleukin-1ß, interleukin-6 and NOX2 were significantly decreased in AR-R17779-treated mice compared with Ang II+HFD mice. AR-R17779 treatment also reduced blood pressure and serum lipid levels. In conclusion, α7nAChR activation attenuates atherogenesis and aortic abdominal aneurysm formation in ApoE-deficient mice possibly through an anti-inflammatory effect and reduction of blood pressure and lipid levels. Pharmacological activation of α7nAChR may have a therapeutic potential against atherosclerotic vascular diseases through multiple mechanisms.

Suppression of abdominal aortic aneurysm formation by inhibition of prolyl hydroxylase domain protein through attenuation of inflammation and extracellular matrix disruption.

In the present study we sought to determine the effect of CoCl2, an inhibitor of PHD (prolyl hydroxylase domain protein), on the development of AAA (abdominal aortic aneurysm). AAA was induced in C57BL/6 mice by periaortic application of CaCl2 (AAA group). NaCl (0.9%)-treated mice were used as a sham control (SHAM group). Mice were treated with 0.05% CoCl2 in the drinking water (AAA/CoCl2 group). At 1 and 6 weeks after the operation, aortic tissue was excised for further examination. After 6 weeks of CaCl2 treatment, aortic diameter and macrophage infiltration into the aortic adventitia were increased in the AAA group compared with the SHAM group. Treatment with CoCl2 reduced the aneurysmal size and macrophage infiltration compared with the AAA group. Aortic expression of inflammatory cytokines and MCP-1 (monocyte chemoattractant protein-1) and the activities of MMP-9 (matrix metalloproteinase-9) and MMP-2 were enhanced in the AAA group and attenuated in the AAA/CoCl2 group. Expression of cytokines and the activities of MMPs were already increased after 1 week of CaCl2 treatment, but were suppressed by CoCl2 treatment in association with reduced NF-κB (nuclear factor κB) phosphorylation. Treatment with CoCl2 in mice prevented the development of CaCl2-induced AAA in association with reduced inflammation and ECM (extracellular matrix) disruption. The results of the present study suggest that PHD plays a critical role in the development of AAA and that there is a therapeutic potential for PHD inhibitors in the prevention of AAA development.

Deletion of phd2 in myeloid lineage attenuates hypertensive cardiovascular remodeling.

BACKGROUND: Hypertension induces cardiovascular hypertrophy and fibrosis. Infiltrated macrophages are critically involved in this process. We recently reported that inhibition of prolyl hydroxylase domain protein 2 (PHD2), which hydroxylates the proline residues of hypoxia-inducible factor-α (HIF-α) and thereby induces HIF-α degradation, suppressed inflammatory responses in macrophages. We examined whether myeloid-specific Phd2 deletion affects hypertension-induced cardiovascular remodeling. METHODS AND RESULTS: Myeloid-specific PHD2-deficient mice (MyPHD2KO) were generated by crossing Phd2-floxed mice with LysM-Cre transgenic mice, resulting in the accumulation of HIF-1α and HIF-2α in macrophage. Eight- to ten-week-old mice were given N(G)-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor, and Angiotensin II (Ang II) infusion. L-NAME/Ang II comparably increased systolic blood pressure in control and MyPHD2KO mice. However, MyPHD2KO mice showed less aortic medial and adventitial thickening, and macrophage infiltration. Cardiac interstitial fibrosis and myocyte hypertrophy were also significantly ameliorated in MyPHD2KO mice. Transforming growth factor-ß and collagen expression were decreased in the aorta and heart from MyPHD2KO mice. Echocardiographic analysis showed that left ventricular hypertrophy and reduced ejection fraction induced by L-NAME/Ang II treatment in control mice were not observed in MyPHD2KO mice. Administration of digoxin that inhibits HIF-α synthesis to L-NAME/Ang II-treated MyPHD2KO mice reversed these beneficial features. CONCLUSIONS: Phd2 deletion in myeloid lineage attenuates hypertensive cardiovascular hypertrophy and fibrosis, which may be mediated by decreased inflammation- and fibrosis-associated gene expression in macrophages. PHD2 in myeloid lineage plays a critical role in hypertensive cardiovascular remodeling.

Prolyl hydroxylase domain protein 2 plays a critical role in diet-induced obesity and glucose intolerance.

BACKGROUND: Recent studies suggest that the oxygen-sensing pathway consisting of transcription factor hypoxia-inducible factor and prolyl hydroxylase domain proteins (PHDs) plays a critical role in glucose metabolism. However, the role of adipocyte PHD in the development of obesity has not been clarified. We examined whether deletion of PHD2, the main oxygen sensor, in adipocytes affects diet-induced obesity and associated metabolic abnormalities. METHODS AND RESULTS: To delete PHD2 in adipocyte, PHD2-floxed mice were crossed with aP2-Cre transgenic mice (Phd2(f/f)/aP2-Cre). Phd2(f/f)/aP2-Cre mice were resistant to high-fat diet-induced obesity (36.7±1.7 versus 44.3±2.0 g in control; P<0.01) and showed better glucose tolerance and homeostasis model assessment-insulin resistance index than control mice (3.6±1.0 versus 11.1±2.1; P<0.01). The weight of white adipose tissue was lighter (epididymal fat, 758±35 versus 1208±507 mg in control; P<0.01) with a reduction in adipocyte size. Macrophage infiltration into white adipose tissue was also alleviated in Phd2(f/f)/aP2-Cre mice. Target genes of hypoxia-inducible factor, including glycolytic enzymes and adiponectin, were upregulated in adipocytes of Phd2(f/f)/aP2-Cre mice. Lipid content was decreased and uncoupling protein-1 expression was increased in brown adipose tissue of Phd2(f/f)/aP2-Cre mice. Knockdown of PHD2 in 3T3L1 adipocytes induced a decrease in the glucose level and an increase in the lactate level in the supernatant with upregulation of glycolytic enzymes and reduced lipid accumulation. CONCLUSIONS: PHD2 in adipose tissue plays a critical role in the development of diet-induced obesity and glucose intolerance. PHD2 might be a novel target molecule for the treatment of obesity and associated metabolic abnormalities.

Beraprost sodium, a stable prostacyclin analogue, improves insulin resistance in high-fat diet-induced obese mice.

Obesity induces hypertrophy of adipocyte resulting in production of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and monocyte chemoattractant protein 1 (MCP1 (CCL2)). These cytokines play an important role in the development of insulin resistance. Beraprost sodium (BPS), a prostaglandin I2 analogue, is reported to attenuate inflammation. In this study, we examined the effect of BPS on glucose metabolism in mice fed a high-fat diet (HFD). Four-week-old C57/B6 male mice were fed a HFD for 12 weeks (HFD group) and the treatment group received oral BPS (300 µg/kg per day) for the same period. Then, glucose metabolism, histological changes, and gene expression of white adipose tissue (WAT) were examined. Body weight was increased, and glucose intolerance and insulin resistance were developed in the HFD group. Treatment with BPS improved glucose tolerance and insulin action without body weight change. Histological analysis of WAT showed an increase in the size of adipocyte and macrophage infiltration in the HFD group, which was attenuated by BPS treatment. BPS reduced HFD-induced expression of MCP1 and TNF-α in WAT. BPS also attenuated hepatic steatosis induced by the HFD. These results suggest that BPS improved glucose intolerance possibly through suppression of inflammatory cytokines in WAT. BPS may be beneficial for the treatment of obesity-associated glucose intolerance.