Shogo Imagawa

Interleukin-6 and Mevastatin Regulate Plasminogen Activator Inhibitor-1 Through CCAAT/Enhancer-Binding Protein-δ

Objective—We sought to determine the etiologic mechanism of proinflammatory cytokine, interleukin-6 (IL-6), and statin as regulators of synthesis of plasminogen activator inhibitor-1 (PAI-1), the physiological fibrinolysis inhibitor and an acute-phase reactant. Methods and Results—Transient transfection and luciferase assay in HepG2 human hepatoma-derived cells demonstrated that IL-6 increased PAI-1 promoter activity and mevastatin decreased IL-6–inducible response. Systematic deletion assay of the promoter demonstrated that the region (−239 to −210 bp) containing a putative CCAAT/enhancer-binding protein (C/EBP) binding site was necessary. Point mutation in this site abolished the IL-6–inducible response. Electrophoretic mobility shift assay and chromatin immunoprecipitation assay demonstrated that C/EBPα, C/EBPβ, and C/EBP&dgr; were involved in protein–DNA complex formation in intact cells. Deoxyribonuclease (DNase) I footprinting analysis revealed that 5′ flanking region (−232 to −210 bp) is acute-phase response protein-binding site. C/EBP&dgr; binding activity was increased by IL-6 and attenuated by mevastatin. Mevastatin attenuated IL-6–mediated increase of C/EBP&dgr; protein in the nuclear extracts. IL-6 also increased PAI-1 and C/EBP&dgr; mRNA in mouse primary hepatocytes. Conclusions—IL-6 increases hepatic PAI-1 expression mediated by the −232- to −210-bp region of the promoter containing a C/EBP&dgr; binding site. Vascular protection by statins may be partly mediated through regulation of CEBP&dgr; and consequent modulation of PAI-1 expression.

Hepatocyte Growth Factor Regulates E Box–Dependent Plasminogen Activator Inhibitor Type 1 Gene Expression in HepG2 Liver Cells

Objective—We sought to determine the etiologic mechanism of pleiotropic growth factor, hepatocyte growth factor (HGF), as a regulator of hepatic synthesis of plasminogen activator inhibitor (PAI)-1, the physiological inhibitor of fibrinolysis and a potential inducer of atherothrombosis. Methods and Results—HGF increased PAI-1 mRNA expression and PAI-1 protein accumulation in the conditioned media of human liver-derived HepG2 cells, and increased hepatic PAI-1 mRNA expression in vivo in mice. HGF-inducible PAI-1 mRNA was attenuated by U0126, a specific inhibitor of mitogen-activated protein kinase (MAPK) kinase, and genistein, an inhibitor of tyrosine kinase. HGF increased the human PAI-1 promoter (−829 to +36 bp) activity, and deletion and mutation analysis uncovered a functional E box (5′-CACATG-3′) at positions −158 to −153 bp. Electrophoretic mobility shift assays demonstrated that this E box binds upstream stimulatory factors (USFs). HGF phosphorylated USFs through MAPK and tyrosine kinase pathways. Co-transfection of USF1 expression vector increased PAI-1 promoter activity. Sterol regulatory element-binding protein-1 attenuated HGF-inducible PAI-1 promoter activity. Conclusions—Because USFs are involved in the regulation of carbohydrates and lipid metabolism, HGF-mediated PAI-1 production may provide a novel link between atherothrombosis and metabolic derangements. Targeting HGF signaling pathway may modulate the thrombotic risk in high-risk patients.

Increased plasma concentrations of N-terminal pro-brain natriuretic peptide reflect the presence of mildly reduced left ventricular diastolic function in hypertension.

BackgroundThe potential use of assays of N-terminal pro-brain natriuretic peptide for detection of diastolic abnormalities associated with alterations in blood pressure has not been elucidated. This study was designed to determine whether increased plasma concentrations of N-terminal pro-brain natriuretic peptide sensitively reflect abnormal diastolic function associated with hypertension. MethodsConcentrations of N-terminal pro-brain natriuretic peptide in plasma were assayed in 40 previously untreated hypertensive patients without overt congestive heart failure and in 20 age and sex-matched controls. Hypertensive patients were studied with the use of pulsed Doppler and color M-mode Doppler echocardiography for the evaluation of left ventricular diastolic function. ResultsConcentrations of N-terminal pro-brain natriuretic peptide were elevated in hypertensive patients [75.1±75.2 (SD) pg/ml compared with 37.9±38.5 in controls, P<0.05]. In hypertensive patients, concentrations of N-terminal pro-brain natriuretic peptide were negatively correlated with the ratio of color M-mode flow propagation velocity to transmitral E velocity consistent with the view that increased concentrations of N-terminal pro-brain natriuretic peptide are indicative of alterations in diastolic function. Hypertensive patients with N-terminal pro-brain natriuretic peptide values above the mean value in the control group exhibited significantly increased brachial intimal–medial thickness and reduced wall stress, consistent with the view that increased N-terminal pro-brain natriuretic peptide was associated with favorable peripheral arterial remodeling. ConclusionsElevated concentrations of N-terminal pro-brain natriuretic peptide in plasma reflect the presence of left ventricular diastolic abnormalities and peripheral arterial remodeling in asymptomatic patients with hypertension.

Impact of statins on modulation by insulin of expression of plasminogen activator inhibitor type-1.

ObjectivePlasminogen activator inhibitor type-1 (PAI-1) is a physiologic inhibitor of fibrinolysis and a known determinant of cardiovascular risk. That its expression is stimulated by insulin in HepG2 human hepatoma cells has been shown earlier. Others have found that increased expression of PAI-1 is a risk factor for acute myocardial infarction. Pleiotropic (noncholesterol lowering) effects of statins seem to reduce cardiovascular risk. This study was designed to determine whether insulin stimulation of PAI-1 expression is attenuated by statins, and if so to explore mechanisms that are responsible for the attenuation. MethodsPAI-1 protein in the conditioned media was assayed by western blotting, and PAI-1 mRNA expression was measured by real-time reverse transcriptase polymerase chain reactions. ResultsInsulin (0.001–10 μmol/l) increased accumulation of PAI-1 protein in the conditioned media and PAI-1 mRNA expression in HepG2 cells. A transient transfection assay of the human PAI-1 promoter-luciferase construct demonstrated that insulin increased PAI-1 promoter activity. Increased PAI-1 mRNA was attenuated significantly by U0126 and PD98059, specific inhibitors of mitogen-activated protein kinase. In contrast, GF109203X, an inhibitor of the protein kinase C pathway, and LY294002, an inhibitor of phosphatidylinositol 3-kinase, exerted no effects. Simvastatin (10 μmol/l), known to translocate membrane-bound sterol regulatory element-binding protein to nuclei, attenuated PAI-1 mRNA expression induced by insulin. It did not affect baseline PAI-1 mRNA expression. Intraperitoneal injection of insulin (0.1 IU/kg) increased concentrations of PAI-1 antigen in plasma in mice within 3 h, correlating with hepatic PAI-1 mRNA expression. ConclusionInsulin-mediated augmented expression of PAI-1 may be amenable to suppression attributable to pleiotropic effects of statins, potentially diminishing cardiovascular risk in patients with insulin resistance.

Intracellular signal transduction modulating expression of plasminogen activator inhibitor-1 in adipocytes.

The concentrations in blood of plasminogen activator inhibitor-1 (PAI-1), an inhibitor of fibrinolysis and proteolysis, are elevated in obese and insulin-resistant subjects, predispose them to the risk of thrombosis, and may accelerate atherogenesis. Adipose tissue is a prominent source. Accordingly, intracellular signaling pathways that may influence PAI-1 expression in adipocytes have been the focus of considerable study. Rho, a small GTP binding and GTPase protein, when activated in turn activates its target, Rho-associated coiled-coil forming protein, to yield an active kinase, Rho-kinase, an effector in the Rho pathway. Rho-kinase exerts calcium-sensitizing effects in vascular smooth muscle cells and inhibitory effects on transforming growth factor-beta (TGF-beta) expression in chicken embryonic heart cells. Because TGF-beta is a powerful agonist of PAI-1 expression, we characterized the effects of inhibition of Rho-kinase in 3T3-L1 adipocytes. PAI-1 mRNA was determined by Northern blotting, and PAI-1 protein was determined by Western blotting. The Rho-kinase inhibitor, Y-27632 [(R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide], increased PAI-1 expression markedly. Although genistein, a flavonoid tyrosine kinase, attenuated the increase of PAI-1 induced by Y-27632, other non-flavonoid tyrosine kinase inhibitors did not. However, another flavonoid, daidzein, which lacks tyrosine kinase activity, decreased basal PAI-1 expression and attenuated the induction of PAI-1 expression by Y-27632. Thus, the Rho/Rho-kinase system inhibits PAI-1 expression by a flavonoid-sensitive mechanism in adipocytes. Therefore, flavonoids may be useful in decreasing elevated PAI-1 expression in adipose tissue and its consequent pathophysiologic sequelae.

Coronary capillary network remodeling and hypofibrinolysis in aged obese diabetic rats: Implications for increased myocardial vulnerability to ischemia

Despite the known abnormalities of cardiac function in patients with overt non-insulin dependent diabetes mellitus (NIDDM) the temporal changes of coronary capillary network remodeling leading to potential microcirculatory dysfunction have not been elucidated. To this end, left ventricular subendocardial capillary network of Otsuka Long-Evans Tokushima Fatty (OLETF) rats, characterized by hypertension, obesity, hyperglycemia, hyperinsulinemia and mild NIDDM, and control Long-Evans Tokushima (LETO) rats were investigated. Total capillary density in OLETF was significantly higher than that in LETO at 20 weeks, suggesting compensatory improvement of O2 transport at early stages of NIDDM. The increase in capillary density in OLETF was lost at 40 and 60 weeks due to the decreases of intermediate capillary portions and venular capillary portions. Although capillary domain area (area innervated by single capillary) in OLETF was lower than that in LETO at 20 weeks, the values were similar between OLETF and LETO at 40 and 60 weeks, suggesting that adaptive improvement in the capacity for O2 transport with a high perfusion was lost in late stages of NIDDM. Activity of plasma plasminogen activator inhibitor-1 (PAI-1), the major physiologic inhibitor of proteo(fibrino)lysis, in OLETF was higher than that in LETO at 40 and 60 weeks, suggesting that increase of PAI-1 may downregulate compensatory adaptive capillary network remodeling by inhibiting proteolysis and angiogenesis in the cardiac interstitium. Loss of adaptive myocardial microcirculation may therefore contribute to increased vulnerability in ischemic injury and to cardiac dysfunction in NIDDM.

Left ventricular noncompaction with intractable heart failure responsive to empagliflozin

Despite several reports on the mechanism of the effect of empagliflozin, which has the potential for improved prognosis in heart failure, it is still not fully understood. We experienced a case of left ventricular noncompaction that caused fluid retention in a patient who showed resistance to existing diuretics. By using empagliflozin, we successfully treated this case of acute heart failure and observed stabilized symptoms with no renal dysfunction and deterioration of patient condition. Although the potential for improved prognosis with this drug in a high-risk group for cardiovascular events has been reported, based on EMPA-REG OUTCOME trial results, there are few reports on its effect of treatment and mechanism in treating acute heart failure. The effect of this drug in treating heart failure from the acute phase to the chronic phase can be expected. <Learning objective: Empagliflozin is a drug that works from the acute phase and is effective from the acute phase to the chronic phase, in treating intractable heart failures that demonstrate resistance to existing drug treatments.>.