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Biomedical subjects

Keisuke Fukuo

Publications and source records attributed to Keisuke Fukuo.

13 recordsLinked to original sources
PubMed2006

Apop-1, a novel protein inducing cyclophilin D-dependent but Bax/Bak-related channel-independent apoptosis.

In the intrinsic pathway of apoptosis, mitochondria play a crucial role by releasing cytochrome c from the intermembrane space into the cytoplasm. Cytochrome c release through Bax/Bak-dependent channels in mitochondria has been well documented. In contrast, cyclophilin D (CypD), an important component of permeability transition pore-dependent protein release, remains largely undefined, and no apoptogenic proteins that act specifically in a CypD-dependent manner have been reported to date. Here, we describe a novel and evolutionarily conserved protein, apoptogenic protein (Apop). Mouse Apop-1 expression induces apoptotic death by releasing cytochrome c from mitochondria into the cytosolic space followed by activation of caspase-9 and -3. Apop-1-induced apoptosis is not blocked by Bcl-2 or Bcl-xL, inhibitors of Bax/Bak-dependent channels, whereas it is completely blocked by cyclosporin A, an inhibitor of permeability transition pore. Cells lacking CypD were resistant to Apop-induced apoptosis. Moreover, inhibition of Apop expression prevented the cell death induced by apoptosis-inducing substances. Our findings, thus, indicate that the expression of Apop-1 induces apoptosis though CypD-dependent pathway and that Apop-1 plays roles in cell death under physiological conditions.

Amino Acid Sequence↗
PubMed2006

Fas ligand mRNA levels of circulating leukocytes reflect endothelial dysfunction in hyperlipidemic but not in non-hyperlipidemic patients.

To find a novel marker for identifying patients at high-risk for endothelial dysfunction among patients with atherosclerosis, we examined the correlation between mRNA levels of Fas ligand (FasL), an apoptosis-inducing factor, in circulating leukocytes and clinical parameters in these patients. FasL mRNA levels of circulating leukocytes were measured with the TaqMan-PCR method. A negative correlation was observed between brachial artery flow-mediated dilatation (%FMD) and FasL mRNA levels of leukocytes in hyperlipidemic but not in non-hyperlipidemic patients. %FMD was more impaired in patients with a high level of FasL mRNA than in those with a low level of FasL mRNA. Interestingly, the improvement of %FMD by treatment with a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor (simvastatin) was greater in the group showing a decrease in FasL mRNA than in the group with no such decrease. Additionally, simvastatin suppressed the FasL mRNA expression in leukocytes and decreased plasma oxidized low-density lipoprotein (OxLDL) levels. Furthermore, the supernatant of cultured leukocytes from hyperlipidemic patients induced cell death in Jurkat T cells, which was neutralized by an antibody against FasL. These findings suggest that high FasL mRNA expression in circulating leukocytes may be a marker of high-risk for endothelial dysfunction in hyperlipidemic but not in non-hyperlipidemic patients. This information may provide a novel basis for targeting of statin therapy in patients with vulnerable plaques.

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PubMed2006

Tissue inhibitor of metalloproteinase-3 plays important roles in the kidney following unilateral ureteral obstruction.

Tissue inhibitor of metalloproteinase-3 (Timp-3), an inhibitor of matrix-degrading enzymes, is an important molecule for maintenance of the extracellular matrix. In this study, we generated Timp-3-deficient mice and used them to examine the effect of Timp-3-deficiency on blood pressure and to investigate the role of Timp-3 in the kidney following unilateral ureteral obstruction. The blood pressure and heart rate of Timp-3-deficient mice were not significantly different from those of wild-type mice. On the other hand, the obstructed kidneys of Timp-3-deficient mice developed more severe hydronephrosis than those of wild-type animals. Matrix metalloproteinase activities assessed by in situ zymography and transforming growth factor-beta expression were elevated in Timp-3-deficient mice. The renal tissues were thinner and the ratio of renal medulla to cortex was significantly lower in the obstructed Timp-3-deficient kidneys. These findings indicate that Timp-3-deficiency does not substantially affect the blood pressure in mice, and that Timp-3 plays an important role in the maintenance of renal macrostructure after unilateral ureteral obstruction.

Animals↗
PubMed2006

Apoptosis signal-regulating kinase 1 mediates cellular senescence induced by high glucose in endothelial cells.

Vascular ageing is accelerated in patients with diabetes. However, the underlying mechanism remains unclear. Here, we show that high glucose induces activation of apoptosis signal-regulating kinase 1 (ASK1), an apoptosis-inducing signal that mediates endothelial cell senescence induced by hyperglycemia. High glucose induced a time-dependent increase in the levels of ASK1 expression and its activity in human umbilical vein endothelial cells (HUVECs). Incubation of endothelial cells with high glucose increased the proportion of cells expressing senescence-associated beta-galactosidase (SA-beta-gal) activity. However, transfection with an adenoviral construct including a dominant negative form of ASK1 gene significantly inhibited SA-beta-gal activity induced by high glucose. In addition, infection with an adenoviral construct expressing the constitutively active ASK1 gene directly induced an increase in the levels of SA-beta-gal activity. Activation of the ASK1 signal also enhanced plasminogen activator inhibitor-1 (PAI-1) expression in HUVECs. Induction of senescent endothelial cells in aortas and elevation of plasma PAI-1 levels were observed in streptozotocin (STZ) diabetic mice, whereas these changes induced by STZ were attenuated in ASK1-knockout mice. Our results suggest that hyperglycemia accelerates endothelial cell senescence and upregulation of PAI-1 expression through activation of the ASK1 signal. Thus, ASK1 may be a new therapeutic target to prevent vascular ageing and thrombosis in diabetic patients.

Adenoviridae↗
PubMed2004

Homocysteine enhances endothelial apoptosis via upregulation of Fas-mediated pathways.

Hyperhomocysteinemia is an independent risk factor for the development of atherosclerosis. However, the underlying mechanism of endothelial cell injury in hyperhomocysteinemia has not been elucidated. In this study, we examined the effect of homocysteine (Hcy) on Fas-mediated apoptosis in endothelial cells. Hcy-induced upregulation of Fas in endothelial cells (ECs) in a dose-dependent manner. At the same time, Hcy increased intracellular peroxide in ECs. Hcy-induced Fas expression was inhibited by the treatment with catalase. Hcy increased NF-kappaB DNA binding activity, and adenovirus-mediated transfection of a Ikappa-B mutant (Ikappa-B mt) gene inhibited Hcy-induced Fas expression. ECs were sensitive to Fas-mediated apoptosis when exposed to Hcy. Under these condition, Ikappa-B mt protected ECs from Fas-mediated apoptosis. In addition, Hcy inhibited expression of the caspase-8 inhibitor FLICE-inhibitory protein (FLIP). Adenovirus-mediated transfection of constitutively active Akt gene abolished the Hcy-mediated downregulation of FLIP. These data suggest that upregulation of Fas expression and downregulation of FLIP is a mechanism through which Hcy induces EC apoptosis.

Animals↗
PubMed2004

Fas signaling induces Akt activation and upregulation of endothelial nitric oxide synthase expression.

A growing body of evidence has shown that Fas, a death receptor, mediates apoptosis-unrelated biological effects. Here, we report that Fas engagement with Fas ligand induced activation of Akt and upregulation of endothelial nitric oxide synthase expression without induction of apoptosis. In the presence of the phosphatidylinositol 3-kinase inhibitor wortmannin, Fas ligand, however, induced apoptosis instead of upregulation of endothelial nitric oxide synthase expression. In vivo, systolic blood pressure was slightly higher in mutant mice with decreased cell surface Fas expression (lpr mice) compared with wild-type mice. In addition, chronic inhibition of nitric oxide synthesis by N(G)-nitro-l-arginine induced a progressive increase in the levels of blood pressure in wild-type mice, whereas no further increase in the levels of blood pressure was observed in lpr mice. Furthermore, acetylcholine caused a lesser endothelium-dependent relaxation of the strips from lpr mice compared with wild-type mice, although the vasoconstrictor potency of phenylephrine was not different between the two groups. These findings indicate that Fas signaling may have a role in the regulation of endothelial function and blood pressure through modulating endothelial nitric oxide synthase expression in the Akt signal-dependent manner.

Acetylcholine↗
PubMed2003

Eicosapentaenoic acid protects endothelial cells against anoikis through restoration of cFLIP.

Dietary supplementation with eicosapentaenoic acid (EPA) improves the prognosis of chronic inflammatory diseases, including atherosclerosis. The mechanism underlying these beneficial effects, however, remains to be elucidated. Here we show that EPA protects endothelial cells from anoikis through upregulation of the cellular FLICE (Fas-associating protein with death domain-like interleukin-1-converting enzyme)-inhibitory protein (cFLIP), an endogenous inhibitor of caspase-8. EPA-induced upregulation of cFLIP expression was partially suppressed by the phosphatidylinositol-3-kinase inhibitor wortmannin. Conversely, treatment with insulinlike growth factor-1 (IGF-1), an activator of phosphatidylinositol-3-kinase/Akt signaling, or infection with an adenoviral construct expressing the constitutively active Akt gene induced upregulation of cFLIP expression. In addition, pretreatment of endothelial cells with either EPA or IGF-1 protected them from anoikis, suggesting that EPA-induced protection against anoikis is partially mediated through activation of Akt. On the other hand, when endothelial cells were already detached, treatment of these cells with EPA but not with IGF-1 protected them against anoikis. Importantly, EPA restored cFLIP expression without activating Akt signaling in detached endothelial cells, whereas IGF-1 had no effect. Additionally, exogenously restored expression of cFLIP by the tetracycline-regulated adenovirus system protected endothelial cells against anoikis. Furthermore, EPA was protective against the loss of endothelium in an organ culture of rat aortas. These findings suggest that EPA protects against endothelial cell anoikis through restoration of cFLIP expression, which might contribute to the mechanism underlying the beneficial effects of EPA in patients with hypertension.

Anoikis↗
PubMed2003

Upregulation of cAMP is a new functional signal pathway of Klotho in endothelial cells.

We measured angiotensin I-converting enzyme (ACE) activity in a human endothelial cell to characterize the intracellular signal pathways of Klotho. COS-1 cells transfected with naked mouse membrane-form klotho plasmid DNA (pCAGGS-klotho) translated proper Klotho protein. This translated Klotho protein was secreted into the culture medium. Furthermore, ACE activity in human umbilical vein endothelial cells (HUVEC) was upregulated when HUVEC were co-cultured with COS-1 cells that were pre-transfected with pCAGGS-klotho. The conditioned medium from COS-1 cells pre-transfected with pCAGGS-klotho also dose-dependently upregulated ACE in HUVEC. In addition, the conditioned medium induced time- and dose-dependent enhancement of cAMP production in HUVEC. Rp-cAMP, an inhibitor of cAMP-dependent protein kinase A (PKA), inhibited the upregulation of ACE by Klotho protein. Our results suggest that mouse membrane-form Klotho protein acts as a humoral factor to increase ACE activity in HUVEC via a cAMP-PKA-dependent pathway. These findings may provide a new insight into the mechanism of Klotho protein.

Animals↗
PubMed2003

Nifedipine upregulates manganese superoxide dismutase expression in vascular smooth muscle cells via endothelial cell-dependent pathways.

Calcium antagonists normalize endothelial dysfunction and improve the clinical outcome in patients with hypertension. However, the mechanism underlying these beneficial effects remains to be elucidated. Here, we show that the calcium antagonist nifedipine upregulates the expression of manganese superoxide dismutase (Mn SOD), an endogenous antioxidant enzyme, in vascular smooth muscle cells (VSMC) via cellular interactions between VSMC and endothelial cells (EC). Nifedipine induced upregulation of Mn SOD activity and expression in VSMC when cocultured with EC but not when cultured individually. NG-Monomethyl-L-arginine (L-NMMA), an inhibitor of nitric oxide (NO) synthesis, inhibited the upregulation of Mn SOD expression induced by nifedipine. Additionally, N-ethyl-2-(1-ethyl-2-hydroxy-2-nitrosohydrazino) ethanamine, a NO donor, reversed this inhibition by L-NMMA, indicating that NO may be involved in the mechanism underlying the nifedipine-induced upregulation of Mn SOD in VSMC. Preincubation of VSMC with Mn SOD antisense oligodeoxyribonucleotides (ODN) blocked the suppressive effects of nifedipine on DNA synthesis in VSMC cocultured with EC, whereas sense ODN had no effect. We conclude that the calcium antagonist nifedipine induces upregulation of Mn SOD expression in VSMC via NO derived from EC. This finding may provide some insight into the mechanism underlying the beneficial effects of calcium antagonists in patients with hypertension.

Blotting, Western↗
PubMed2002

Nifedipine indirectly upregulates superoxide dismutase expression in endothelial cells via vascular smooth muscle cell-dependent pathways.

BACKGROUND: Calcium antagonists normalize endothelial dysfunction in many cardiovascular diseases. There is no known receptor, however, for calcium antagonists in endothelial cells (ECs). We hypothesized that vascular smooth muscle cells (VSMCs) are involved in the mechanism underlying the normalization of endothelial dysfunction by calcium antagonists. METHODS AND RESULTS: Coculture studies with ECs and VSMCs were performed to determine whether VSMCs mediate modulation of endothelial superoxide dismutase (SOD) activity and expression induced by the calcium antagonist nifedipine. Nifedipine induced upregulation of SOD activity in rat aortic segments but had no effect on SOD expression or activity in ECs or VSMCs cultured individually. When ECs were cocultured with VSMCs, however, nifedipine upregulated SOD expression and activity in ECs. Nifedipine stimulated vascular endothelial growth factor (VEGF) production from VSMCs, and this stimulation of VEGF production was abolished by HOE-140, an antagonist of the bradykinin B(2) receptor. A neutralizing antibody against VEGF inhibited the upregulation of endothelial SOD by nifedipine. In addition, recombinant VEGF induced an increase in the levels of SOD expression in ECs, and supernatant derived from nifedipine-treated VSMCs enhanced NO production from ECs. This increase in NO production by the supernatant was inhibited by preincubation of ECs with SOD antisense oligodeoxyribonucleotides. CONCLUSIONS: The calcium antagonist nifedipine indirectly upregulates endothelial SOD expression by stimulating VEGF production from adjacent VSMCs. This finding may provide further insight into the mechanism underlying the beneficial effects of calcium antagonists in cardiovascular diseases.

Animals↗
PubMed2002

Increased plasma levels of the soluble form of Fas ligand in patients with acute myocardial infarction and unstable angina pectoris.

OBJECTIVES: To examine whether the Fas/Fas ligand system is involved in the pathogenesis of acute myocardial infarction (AMI), we measured the levels of the soluble form of the Fas ligand (sFasL) in the plasma of patients with AMI and stable or unstable angina pectoris (AP). BACKGROUND: The Fas ligand (FasL) is rapidly cleaved off by a metalloproteinase from the cell membrane to become a soluble form as a cytokine. Fas is expressed in most cells, including cardiomyocytes, whereas FasL is mainly expressed in inflammatory cells such as macrophages, which are greatly accumulated in unstable plaque. METHODS: Thirty patients with AMI, 10 patients with unstable AP, 10 patients with stable AP and 30 control subjects were enrolled in the present study. RESULTS: Plasma sFasL levels were significantly elevated on hospital admission in patients with AMI and unstable AP, compared with control subjects. Time-course studies revealed that plasma sFasL levels rapidly decreased within 3 h and then increased again after percutaneous transluminal coronary angioplasty in patients with AMI, but not in patients with stable AP. Importantly, the sFasL levels were higher in the coronary sinus than in the circulation. In addition, in vitro studies showed that the expression of FasL messenger ribonucleic acid was upregulated in mononuclear cells isolated from patients with AMI and that hypoxia stimulated the release of sFasL from isolated mononuclear cells. CONCLUSIONS: This demonstration of elevated levels of sFasL in patients with AMI and unstable AP suggests that activation of the Fas/FasL system may play a pathogenic role in AMI and acute coronary syndromes.

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