Koichi Nishiyama

Heightened Tissue Factor Associated With Tissue Factor Pathway Inhibitor and Prognosis in Patients With Unstable Angina

BACKGROUND This study was designed to evaluate the plasma levels of tissue factor (TF) and tissue factor pathway inhibitor (TFPI) in patients with unstable angina and investigate whether there is a relationship between these levels and unfavorable outcome. METHODS AND RESULTS The plasma TF and free TFPI antigen levels were determined in plasma samples taken from 51 patients with unstable angina, 56 with stable exertional angina, and 55 with chest pain syndrome. The plasma TF and free TFPI antigen levels were higher in the unstable angina group than in the stable exertional angina and chest pain syndrome group. There was a good correlation between TF and TFPI. We established borderline as maximum level in the patients with chest pain syndrome. Seven patients (of the 22 in the high TF group) required revascularization to control their unstable angina during in-hospital stay. On the other hand, only 1 of the 29 patients in the low TF group required myocardial revascularization. Four patients of the 14 patients in the high free TFPI group required myocardial revascularization during in-hospital stay, and 4 of the 37 patients in the low free TFPI group required myocardial revascularization. We compared the TF and free TFPI levels between the cardiac event (+) group and cardiac event (-) group. TF levels were significantly higher in the cardiac event (+) group than in the cardiac event (-) group. CONCLUSIONS We have demonstrated that not only the plasma TF levels but also the plasma-free TFPI levels are elevated in patients with unstable angina. Patients with unstable angina and heightened TF and free TFPI are at increased risk for unfavorable outcomes. The heightened TF level was a more important predictor in patients with unstable angina.

Multiple renal cysts, urinary concentration defects, and pulmonary emphysematous changes in mice lacking TAZ

TAZ (transcriptional coactivator with PDZ-binding motif), also called WWTR1 (WW domain containing transcription regulator 1), is a 14-3-3-binding molecule homologous to Yes-associated protein. TAZ acts as a coactivator for several transcription factors as well as a modulator of membrane-associated PDZ domain-containing proteins, but its (patho)physiological roles remain unknown. Here we show that gene inactivation of TAZ in mice resulted in pathological changes in the kidney and lung that resemble the common human diseases polycystic kidney disease and pulmonary emphysema. Taz-null/lacZ knockin mutant homozygotes demonstrated renal cyst formation as early as embryonic day 15.5 with dilatation of Bowmans capsules and proximal tubules, followed by pelvic dilatation and hydronephrosis. After birth, only one-fifth of TAZ-deficient homozygotes grew to adulthood and demonstrated multicystic kidneys with severe urinary concentrating defects and polyuria. Furthermore, adult TAZ-deficient homozygotes exhibited diffuse emphysematous changes in the lung. Thus TAZ is essential for developmental mechanisms involved in kidney and lung organogenesis, whose disturbance may lead to the pathogenesis of common human diseases.

Comparison of Plasma Tissue Factor Levels in Unstable and Stable Angina Pectoris

We have reported that the plasma levels of plasma fibrinopeptide A and plasminogen activator inhibitor activity increase in patients with unstable angina and acute myocardial infarction. Tissue factor (TF) is a low-molecular-weight glycoprotein that binds to and acts on essential cofactor VII, and the resulting complex activates factors IX and X, initiating the coagulation cascade. We measured plasma TF antigen levels in 21 patients with unstable angina (on admission and after treatment), 27 patients with stable exertional angina, and 27 control subjects. The 3 groups were matched for age, gender, and other clinical variables. The plasma TF antigen levels were higher in the unstable angina group than in the stable exertional angina and control groups (240 +/- 75 vs 184 +/- 46 and 177 +/- 37 pg/ml, p < 0.01). There were no significant differences in the plasma TF antigen levels between the stable exertional angina and the control groups. Furthermore, the plasma TF antigen levels were reexamined after treatment in the 21 patients with unstable angina. The mean level in these 21 patients decreased after 2 weeks of treatment (from 240 +/- 75 to 206 +/- 57 pg/ml, p < 0.01). This study suggests that the plasma TF antigen levels correlate with disease activity in patients with unstable angina. The increased plasma TF antigen levels in patients with unstable angina may reflect intravascular procoagulant activity.

AMP-activated Protein Kinase Activation Increases Phosphorylation of Glycogen Synthase Kinase 3β and Thereby Reduces cAMP-responsive Element Transcriptional Activity and Phosphoenolpyruvate Carboxykinase C Gene Expression in the Liver

AMP-activated protein kinase (AMPK) activation reportedly suppresses transcriptional activity of the cAMP-responsive element (CRE) in the phosphoenolpyruvate carboxykinase C (PEPCK-C) promoter and reduces hepatic PEPCK-C expression. Although a previous study found TORC2 phosphorylation to be involved in the suppression of AMPK-mediated CRE transcriptional activity, we herein present evidence that glycogen synthase kinase 3β (GSK3β) phosphorylation induced by AMPK also plays an important role. We initially found that injecting fasted mice with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) markedly increased Ser-9 phosphorylation of hepatic GSK3β within 15 min. Stimulation with AICAR or the GSK3β inhibitor SB-415286 strongly inhibited CRE-containing promoter activity in HepG2 cells. Using the Gal4-based transactivation assay system, the transcriptional activity of cAMP-response element-binding protein (CREB) was suppressed by both AICAR and SB415286, whereas that of TORC2 was repressed significantly by AICAR but very slightly by SB415286. These results show inactivation of GSK3β to directly inhibit CREB but not TORC2. Importantly, the AICAR-induced suppression of PEPCK-C expression was shown to be blunted by overexpression of GSK3β(S9G) but not wild-type GSK3β. In addition, AICAR stimulation decreased, whereas Compound C (AMPK inhibitor) increased CREB phosphorylation (Ser-129) in HepG2 cells. The time-courses of decreased CREB phosphorylation (Ser-129) and increased GSK3β phosphorylation were very similar. Furthermore, AMPK-mediated GSK3β phosphorylation was inhibited by an Akt-specific inhibitor in HepG2 cells, suggesting involvement of the Akt pathway. In summary, phosphorylation (Ser-9) of GSK3β is very likely to be critical for AMPK-mediated PEPCK-C gene suppression. Reduced CREB phosphorylation (Ser-129) associated with inactivation of GSK3β by Ser-9 phosphorylation may be the major mechanism underlying PEPCK-C gene suppression by AMPK-activating agents such as biguanide.

Angiotensin-converting enzyme inhibition reduces monocyte chemoattractant protein-1 and tissue factor levels in patients with myocardial infarction ☆

OBJECTIVES We investigated the effects of enalapril therapy on plasma tissue factor (TF), tissue factor pathway inhibitor (TFPI) and monocyte chemoattractant protein-1 (MCP-1) levels in patients with acute myocardial infarction. BACKGROUND Macrophages express TF in human coronary atherosclerotic plaques. Both TF and TFPI are major regulators of coagulation and thrombosis. Monocyte chemoattractant protein-1 is a monocyte and macrophage chemotactic and activating factor. METHODS In a randomized, double-blind, placebo-controlled study beginning about two weeks after myocardial infarction, 16 patients received four weeks of placebo (placebo group) and another 16 patients received four weeks of enalapril 5 mg daily therapy (enalapril group). We performed blood sampling after administration of the doses. RESULTS There were no significant differences in the serum angiotensin-converting enzyme (ACE) activity, plasma TF, free TFPI or MCP-1 levels before administration between the enalapril and placebo groups. In the enalapril group, ACE activity (IU/liter) (14.0 before, 5.2 on day 3, 5.8 on day 7, 6.3 on day 28), TF levels (pg/ml) (223, 203, 182, 178) and MCP-1 levels (pg/ml) (919, 789, 790, 803) significantly decreased by day 28. However, the free TFPI levels (ng/ml) (28.2, 26.5, 26.8, 28.4) did not change. These four variables were unchanged during the study period in the placebo group. CONCLUSIONS This study demonstrated that administration of enalapril reduces the increased procoagulant activity in patients with myocardial infarction associated with inhibition of the activation and accumulation of macrophages and monocytes.

Angiogenic morphogenesis driven by dynamic and heterogeneous collective endothelial cell movement

Angiogenesis is a complex process, which is accomplished by reiteration of modules such as sprouting, elongation and bifurcation, that configures branching vascular networks. However, details of the individual and collective behaviors of vascular endothelial cells (ECs) during angiogenic morphogenesis remain largely unknown. Herein, we established a time-lapse imaging and computer-assisted analysis system that quantitatively characterizes behaviors in sprouting angiogenesis. Surprisingly, ECs moved backwards and forwards, overtaking each other even at the tip, showing an unknown mode of collective cell movement with dynamic ‘cell-mixing’. Mosaic analysis, which enabled us to monitor the behavior of individual cells in a multicellular structure, confirmed the ‘cell-mixing’ phenomenon of ECs that occurs at the whole-cell level. Furthermore, an in vivo EC-tracking analysis revealed evidence of cell-mixing and overtaking at the tip in developing murine retinal vessels. In parametrical analysis, VEGF enhanced tip cell behavior and directed EC migration at the stalk during branch elongation. These movements were counter-regulated by EC-EC interplay via γ-secretase-dependent Dll4-Notch signaling, and might be promoted by EC-mural cell interplay. Finally, multiple regression analysis showed that these molecule-mediated tip cell behaviors and directed EC migration contributed to effective branch elongation. Taken together, our findings provide new insights into the individual and collective EC movements driving angiogenic morphogenesis. The methodology used for this analysis might serve to bridge the gap in our understanding between individual cell behavior and branching morphogenesis.

Sirt3 protects in vitro–fertilized mouse preimplantation embryos against oxidative stress–induced p53-mediated developmental arrest

Sirtuins are a phylogenetically conserved NAD+-dependent protein deacetylase/ADP-ribosyltransferase family implicated in diverse biological processes. Several family members localize to mitochondria, the function of which is thought to determine the developmental potential of preimplantation embryos. We have therefore characterized the role of sirtuins in mouse preimplantation development under in vitro culture conditions. All sirtuin members were expressed in eggs, and their expression gradually decreased until the blastocyst stage. Treatment with sirtuin inhibitors resulted in increased intracellular ROS levels and decreased blastocyst formation. These effects were recapitulated by siRNA-induced knockdown of Sirt3, which is involved in mitochondrial energy metabolism, and in Sirt3-/- embryos. The antioxidant N-acetyl-L-cysteine and low-oxygen conditions rescued these adverse effects. When Sirt3-knockdown embryos were transferred to pseudopregnant mice after long-term culture, implantation and fetal growth rates were decreased, indicating that Sirt3-knockdown embryos were sensitive to in vitro conditions and that the effect was long lasting. Further experiments revealed that maternally derived Sirt3 was critical. Sirt3 inactivation increased mitochondrial ROS production, leading to p53 upregulation and changes in downstream gene expression. The inactivation of p53 improved the developmental outcome of Sirt3-knockdown embryos, indicating that the ROS-p53 pathway was responsible for the developmental defects. These results indicate that Sirt3 plays a protective role in preimplantation embryos against stress conditions during in vitro fertilization and culture.

Serial Changes in Plasma Levels of Soluble P-Selectin in Patients With Acute Myocardial Infarction ☆

The present study examines whether an acute inflammatory response occurs during acute myocardial infarction (AMI) by measuring soluble P-selectin levels. We examined plasma soluble P-selectin levels in 16 consecutive patients with AMI, in 15 patients with angina, and in 13 control subjects with chest pain but normal coronary arteries and no coronary spasm. In patients with AMI, blood samples were obtained immediately after admission and at 1, 4, 24, and 48 hours, and 1 week after initiation of reperfusion therapy. The plasma soluble P-selectin levels were significantly higher in the AMI group on admission than in the other 2 groups (83 +/- 13 ng/ml, p < 0.01). The plasma soluble P-selectin levels at baseline were not significantly different between the angina and control groups (28 +/- 4 vs 24 +/- 5 ng/ml, p = NS). Plasma soluble P-selectin levels reached their peak significantly at 4 hours after initiation of the reperfusion therapy in patients with AMI. The peak level was significantly higher than the level on admission (115 +/- 17 vs 83 +/- 13 ng/ml, p < 0.05). The plasma soluble P-selectin levels were higher in the AMI group than in the angina and control groups over the time course (p < 0.01). Our data indicate that the plasma soluble P-selectin levels are increased in patients with AMI, and that the levels are increases after reperfusion therapy more than before reperfusion. We suggest that the increase in the plasma soluble P-selectin levels may be caused by the activation of endothelial cells and platelets after myocardial ischemia and reperfusion during AMI.

Resistin-like Molecule β Activates MAPKs, Suppresses Insulin Signaling in Hepatocytes, and Induces Diabetes, Hyperlipidemia, and Fatty Liver in Transgenic Mice on a High Fat Diet

Resistin and resistin-like molecules (RELMs) are a family of proteins reportedly related to insulin resistance and inflammation. Because the serum concentration and intestinal expression level of RELMβ were elevated in insulin-resistant rodent models, in this study we investigated the effect of RELMβ on insulin signaling and metabolism using transgenic mice and primary cultured hepatocytes. First, transgenic mice with hepatic RELMβ overexpression were shown to exhibit significant hyperglycemia, hyperlipidemia, fatty liver, and pancreatic islet enlargement when fed a high fat diet. Hyperinsulinemic glucose clamp showed a decreased glucose infusion rate due to increased hepatic glucose production. In addition, the expression levels of IRS-1 and IRS-2 proteins as well as the degrees of insulin-induced phosphatidylinositol 3-kinase and Akt activations were attenuated in RELMβ transgenic mice. Similar down-regulations of IRS-1 and IRS-2 proteins were observed in primary cultured hepatocytes chronically treated (for 24 h) with RELMβ, suggesting the insulin resistance-inducing effect of RELMβ to be direct. Furthermore, it was shown that RELMβ acutely and markedly activates ERK and p38, while weakly activating JNK, in primary cultured hepatocytes. This increased basal p38 phosphorylation level was also observed in the livers of RELMβ transgenic mice. In conclusion, RELMβ, a gut-derived hormone, impairs insulin signaling probably via the activations of classic MAPKs, and increased expression of RELMβ may be involved in the pathogenesis of glucose intolerance and hyperlipidemia in some insulin-resistant models. Thus, RELMβ is a potentially useful marker for assessing insulin resistance and may also be a target for future novel anti-diabetic agents.

Id1 Gene Transfer Confers Angiogenic Property on Fully Differentiated Endothelial Cells and Contributes to Therapeutic Angiogenesis

Background— Transplantation of endothelial progenitor cells has been proposed as a potential strategy for therapeutic revascularization. However, the limited endogenous cell pool and the related technical difficulties constitute clinically important disadvantages to autologous transplantation. In this study we investigated whether fully differentiated endothelial cells (ECs) modified with gene transfer of Id1, a helix-loop-helix transcription factor involved in angiogenesis, have the potential to contribute to therapeutic angiogenesis. Methods and Results— The Id1 gene was transferred into human umbilical vein ECs (HUVECs) via a Sendai virus vector. Id1 stimulated migration, proliferation, and capillary-like tube/cord formation of HUVECs. In addition, Id1 reduced serum deprivation–induced HUVEC apoptosis, as shown by FACS analysis with annexin V and TUNEL staining. Transplantation of Id1-overexpressing HUVECs accelerated recovery of blood flow as evaluated by laser-Doppler perfusion imaging, increased capillary density, and improved the rate of limb salvage compared with the transplantation of control HUVECs. Histochemical analysis revealed that the regenerated vascular networks of limbs transplanted with Id1-overexpressing HUVECs contained numerous HUVECs, some of which were in a proliferative state. Untransfected HUVECs were also incorporated with Id1-transfected HUVECs, suggesting the noncell autonomous effect of Id1. Finally, angiopoietin-1 was upregulated in Id1-overexpressing HUVECs and functionally contributed to the in vitro angiogenic effect of Id1. Conclusions— Id1 gene transfer conferred HUVECs with an angiogenic property, contributing to neovascularization after transplantation into ischemic lesions. Transplantation of Id1-overexpressing mature ECs may serve as a novel and useful strategy for therapeutic angiogenesis.