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Takuzo Hano

Publications and source records attributed to Takuzo Hano.

At least 19 recordsLinked to original sources
PubMed2006

The effect of senescence of endothelial progenitor cells on in-stent restenosis in patients undergoing coronary stenting.

OBJECTIVE: Restenosis after stent implantation is caused by endothelial cell damage and subsequent neointimal formation. The objective of this study is to elucidate the relevance of endothelial progenitor cells (EPCs) in the development of in-stent restenosis in patients undergoing stent implantation. PATIENTS OR MATERIALS: The subjects were 46 patients who underwent coronary stenting. Blood samples were collected at the time of follow-up coronary angiography after coronary stenting. EPCs were isolated from blood samples and cultured. Their phenotypes were confirmed by uptake of acetylated low-density lipoprotein and binding of fluorescein isothiocyanate-labeled Ulex europaeus agglutinin 1 lectin. The number of colony-forming units (CFUs) and the senescent cells, determined by acidic beta-galactosidase staining, was counted. Angiogenic growth factors secreted by EPCs, such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (b-FGF), hepatocyte growth factor (HGF), and macrophage chemoattractant protein (MCP-1) from the culture medium were also measured by enzyme-linked immunosorbent assay. RESULTS: Patients with in-stent restenosis (defined as >40% stenosis, n=16) had a decreased number of CFUs (p<0.05), and increased senescent cells (p<0.05), compared to patients without restenosis (n=30). There was no significant difference of angiogenic growth factors (VEGF, HGF, b-FGF, and MCP-1) secreted by EPCs between the two groups. On multivariate analysis, an increased number of senescent EPCs was the indepen-dent factor associated with in-stent restenosis (OR 1.10, 95% CI 1.01 to 1.20). CONCLUSION: These data suggested that EPCs might be involved in the development of in-stent restenosis.

Aged↗
PubMed2005

Angiotensin II accelerates endothelial progenitor cell senescence through induction of oxidative stress.

OBJECTIVES: Recent studies have revealed an association between coronary risk factors and both the number and function of bone marrow-derived endothelial progenitor cell (EPC). We investigated the effect of angiotensin II (Ang II) on EPC senescence, leading to the impairment of proliferative activity. METHODS AND RESULTS: EPCs were isolated from peripheral blood and characterized. Both reverse transcription (RT)-polymerase chain reaction (PCR) and Western blotting were used to assess gp91phox expression. Immunofluorescence of nitrotyrosine provided evidence of peroxynitrite formation. Our data indicate that Ang II increased the expression of gp91phox mRNA in a dose-dependent manner, which was attenuated by Ang II type 1 (AT1) receptor antagonist valsartan. Similarly, Western blotting revealed that Ang II stimulated an increase in gp91phox, whereas pre-treatment with Valsartan reduced the Ang II-induced expression of gp91phox protein. Valsartan as well as superoxide dismutase (SOD) also inhibited Ang II-induced peroxynitrite formation. The exposure of cultured EPC to Ang II (100 nmol/l) significantly accelerated the rate of senescence compared to a control during 14 days in culture as determined by acidic beta-galactosidase staining. Ang II-induced EPC senescence was significantly inhibited by pre-treatment of either valsartan or SOD (P < 0.01). Because cellular senescence is critically influenced by telomerase, which elongates telomeres, we measured telomerase activity by using PCR-enzyme-linked immunosorbent-based assay. Ang II significantly diminished telomerase activity, although the effect was significantly reduced by pre-treatment with either valsartan or SOD (P < 0.01). We examined whether Ang II-induced EPC senescence translates into an impairment of EPC proliferation. MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenol)-2-(4-sulfophenyl)-2H-tetrazolium] assay disclosed an inhibitory effect of Ang II on EPC proliferation. CONCLUSIONS: Ang II increases gp91phox expression in EPC, which may contribute to oxidative stress, as evidenced by peroxynitrite formation. Ang II accelerates the onset of EPC senescence via increased oxidative stress, which may be related to telomerase inactivation. In addition, Ang II-induced EPC senescence leads to the impairment of proliferative activity.

Angiotensin II↗
PubMed2005

Estrogen reduces endothelial progenitor cell senescence through augmentation of telomerase activity.

BACKGROUND: Recent studies have demonstrated that aging or senescence constitutes a potential limitation to the ability of endothelial progenitor cells (EPCs) to sustain ischemic tissue and repair. Conversely, estrogens have been shown to accelerate recovery of the endothelium after vascular injury. OBJECTIVE: To investigate whether estrogens are able to prevent senescence of EPCs. METHODS AND RESULTS: Human EPCs were isolated from peripheral blood and characterized. After ex-vivo cultivation, the cells became senescent as determined by acidic beta-galactosidase staining. 17beta-estradiol dose-dependently inhibited the onset of EPC senescence in culture. Because cellular senescence is critically influenced by telomerase, which elongates telomeres, we measured telomerase activity using a polymerase chain reaction (PCR)-enzyme-linked immunosorbent assay (ELISA) technique. 17beta-estradiol significantly increased telomerase activity. Interestingly, reverse transcriptase-PCR analysis demonstrated that 17beta-estradiol dose-dependently increased the catalytic subunit, telomerase reverse transcriptase (TERT) - an effect that was significantly inhibited by pharmacological phosphatidylinositol 3-kinase (PI3-K) blockers (either wortmannin or LY294002). Because the expression of TERT is regulated by the PI3-K/Akt pathway, we examined the effect of 17beta-estradiol on Akt activity in EPCs. Immunoblotting analysis revealed that 17beta-estradiol dose-dependently led to phosphorylation and, thus, to activation of Akt in EPCs. We also examined whether the protective effect of 17beta-estradiol on EPC senescence translates into the augmentation of mitogenic activity in EPCs. A [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenol)-2-(4-sulfophenyl)-2H-tetrazolium] (MTS) assay demonstrated that the mitogenic potential in EPCs treated with 17beta-estradiol exceeded that in untreated (control) EPCs (P < 0.01). In addition, EPCs released vascular endothelial growth factor (VEGF) protein--an effect that was significantly augmented by 17beta-estradiol. Finally, in a Matrigel assay, EPCs treated with both 17beta-estradiol and VEGF were shown to be more likely to integrate into the network formation than those treated with VEGF alone. CONCLUSION: The inhibition of EPC senescence by estrogen in vitro may improve the functional activity of EPCs in a way that is important for potential cell therapy.

Androstadienes↗
PubMed2005

Endothelial progenitor cell senescence is accelerated in both experimental hypertensive rats and patients with essential hypertension.

OBJECTIVES: Recent studies have revealed an association between coronary risk factors and both the number and function of bone marrow-derived endothelial progenitor cells (EPCs). Although hypertension is an important coronary risk factor, the influence to the EPCs is not fully understood. We investigated the effect of hypertension on EPC senescence. METHODS: Experimental study We investigated the number and senescence of EPCs in spontaneously hypertensive rats (SHR/Izm) and deoxycorticosterone acetate (DOCA)-salt hypertensive rats. EPCs were isolated from peripheral blood of rats and were characterized. EPC senescence was detected by acidic beta-galactosidase staining. In addition, we measured the telomerase activity using polymerase chain reaction-enzyme-linked immunosorbent assay. CLINICAL STUDY: EPCs were isolated from peripheral blood samples in 37 patients with essential hypertension. After ex-vivo cultivation, we detected senescence and measured the telomerase activity. The total severity index of hypertension-induced organ damage was calculated by the summation of each severity index in the classification of hypertension severity by Tokyo University (1984). RESULTS: Experimental study The EPC senescence in SHR/Izm and DOCA-salt hypertensive rats was significantly increased compared with that of control rats. The telomerase activities in SHR/Izm and DOCA-salt hypertensive sensitive rats were also significantly lowered compared with those of control rats. Clinical study Compared with the control group, EPCs from hypertensive patients showed accelerated senescence and also showed reduced telomerase activity. In hypertensive patients, the degree of hypertension-induced organ damage was negatively correlated with telomerase activity, and was positively correlated with EPC senescence. CONCLUSIONS: EPC senescence is accelerated in both experimental hypertensive rats and patients with essential hypertension, which may be related to telomerase inactivation. The hypertension-induced EPC senescence may affect the process of vascular remodeling.

Aged↗
PubMed2005

Estrogen reduces angiotensin II-induced acceleration of senescence in endothelial progenitor cells.

The interaction among estrogen, angiotensin II (Ang II), and oxidative stress in endothelial progenitor cells (EPCs) remains unknown. We therefore investigated the potential effect of estrogen on Ang II-induced EPC oxidative stress and senescence in EPCs. EPCs were isolated from peripheral blood and characterized. Both reverse transcription (RT)-polymerase chain reaction (PCR) and Western blotting were used to assess gp91phox and angiotensin type 1 receptor (AT1R) expression. Immunofluorescence of nitrotyrosine provided evidence of peroxynitrite formation. Our data indicate that Ang II increased the expression of gp91phox mRNA and protein, and these effects were attenuated by 17beta-estradiol (E2). The exposure of cultured EPCs to Ang II (100 nmol/l) significantly accelerated the rate of senescence compared to that in control cells during 14 days in culture as determined by acidic beta-galactosidase staining, and this effect was significantly inhibited by E2 (p < 0.01). Because cellular senescence is critically influenced by telomerase, which elongates telomeres, we measured telomerase activity by using a PCR-ELISA-based assay. Ang II significantly diminished telomerase activity, although the effect was significantly reduced by pre-treatment with E2 (p < 0.01). Because we previously demonstrated that both the up-regulation of gp91phox and the acceleration of cellular senescence in Ang II-stimulated EPCs could be abolished by pre-treatment with the AT1R- specific antagonist, valsartan, we also explored the effect of estrogen on AT1R expression. Ang II increased AT1R mRNA and protein expression, and these increases were prevented by E2, suggesting that AT1R may at least partially mediate the inhibitory effect of E2 on Ang II-induced acceleration of senescence in EPCs. In conclusion, estrogen reduces Ang II-induced acceleration of senescence in EPCs partially through down-regulation of AT1R expression.

Angiotensin II↗
PubMed2005

Effect of estrogen on differentiation and senescence in endothelial progenitor cells derived from bone marrow in spontaneously hypertensive rats.

The functional impairment associated with atherogenic factors, including hypertension, constitutes a limitation to the ability of endothelial progenitor cells (EPCs) to repair. In addition, estrogens have been shown to play a role in reendothelialization after vascular injury. We investigated the effects of estrogens on differentiation and senescence of EPCs derived from bone marrow (BM-EPCs) in spontaneously hypertensive rats (SHR/Izm). Bone marrow (BM) cells were obtained from the tibias and femurs of age-matched, male SHR/Izm and Wistar-Kyoto rats (WKY/Izm). The number of differentiated, adherent BM-EPCs derived from SHR/Izm was significantly smaller than the number derived from WKY/Izm. 17beta-Estradiol (E2) significantly increased the number of adherent BM-EPCs from SHR/Izm, and this effect was significantly attenuated by pharmacological phosphatidylinositol 3-kinase (PI3-K) blockers. Immunoblotting analysis revealed that E2 treatment led to phosphorylation of Akt. Senescence, as assessed by acidic beta-galactosidase staining, occurred at a significantly greater rate in the BM-EPCs from SHR/Izm than in those from WKY/Izm, but E2 treatment dramatically delayed the senescence of BM-EPCs from SHR/Izm. A polymerase chain reaction (PCR)-ELISA based assay revealed that telomerase activity in BM-EPCs from SHR/Izm was significantly lower than in those from WKY/Izm, but that E2 treatment significantly augmented it. Both MTS and colony forming unit assay revealed that E2 treatment significantly augmented the functional activity in BM-endothelial cell (EC)-like cells from SHR/Izm compared to that in control BM-EC-like cells (no treatment). In conclusion, the differentiation of BM-EPCs derived from SHR/Izm was significantly decreased compared with that of BM-EPCs from WKY/Izm. In addition, the rate of senescence was significantly greater in the BM-EPCs from SHR/Izm than in those from WKY/Izm. Estrogen was shown to augment differentiation and delay the onset of senescence in BM-EPCs from SHR/Izm.

Animals↗
PubMed2004

Oxidized low-density lipoprotein induces endothelial progenitor cell senescence, leading to cellular dysfunction.

1. Recent studies have revealed an association between coronary risk factors and both the number and function of bone marrow-derived endothelial progenitor cells (EPC). We investigated the effect of oxidized low-density lipoprotein (ox-LDL) on the senescence of EPC, leading to cellular dysfunction. 2. Endothelial progenitor cells were isolated from human peripheral blood and characterized. The exposure of cultured EPC to ox-LDL (10 microg/mL) significantly accelerated the rate of senescence compared with control during 20 days in culture as determined by acidic beta-galactosidase staining. Oxidized LDL-induced EPC senescence was significantly inhibited by pretreatment with either lectin-like ox-LDL receptor-1 (LOX-1) antibody (Ab) or atorvastatin (P < 0.01). 3. Because cellular senescence is critically influenced by telomerase, which elongates telomeres, we measured telomerase activity using a polymerase chain reaction-ELISA-based assay. Oxidized LDL significantly diminished telomerase activity to approximately 50%, an effect that was significantly abolished by pretreatment with either LOX-1 Ab or atorvastatin (P < 0.01). 4. We examined whether ox-LDL-induced EPC senescence translates into EPC dysfunction. An MTS assay disclosed an inhibitory effect of ox-LDL on EPC proliferation. In a Matrigel assay, EPC treated with ox-LDL were less likely to participate in network formation compared with controls. 5. In conclusions, ox-LDL accelerates the onset of EPC senescence, which may be related to telomerase inactivation. Oxidized LDL-induced EPC senescence leads to the impairment of proliferative capacity and network formation.

Atorvastatin↗
PubMed2004

Angiotensin II potentiates vascular endothelial growth factor-induced proliferation and network formation of endothelial progenitor cells.

Bone marrow-derived endothelial progenitor cells (EPCs) in the peripheral blood of adult animals and adult humans have been shown to play a role in neovascularization into neovascular structures. On the other hand, angiotensin II (Ang II) plays a role in the development of many vascular diseases. To investigate whether Ang II affects human vascular endothelial growth factor (VEGF)-induced EPCs proliferation and network formation. Reverse transcription-polymelase chain reaction analysis demonstrated that Ang II induced a significant increase of VEGF receptor kinase domain-containing receptor (KDR) mRNA in a dose- and time-dependent manner; the maximal increase, which was 3-fold the control value, occurred after a 4-h stimulation. In addition, flow cytometric analysis revealed that Ang II up-regulated KDR protein expression in human EPCs. Both the angiotensin type 1 (AT1) receptor antagonist (valsartan: 200 nmol/l) and the PKC inhibitor, bisindolylmaleimide (GFX: 10 micromol/l) reduced Ang II-induced KDR mRNA expression to almost the control level. The culture assay showed that Ang II dose-dependently enhanced VEGF-induced EPC proliferation by activating AT1 receptors, which was also confirmed by the colorimetric MTS assay with the electron coupling reagent mathosulfate. Finally, in a Matrigel assay, EPCs treated with both Ang II and VEGF were shown to be more likely to integrate into the network formation than those treated with VEGF alone. In conclusion, our data indicate that Ang II potentiates VEGF-induced human EPCs proliferation and network formation through the up-regulation of KDR.

Angiotensin II↗
PubMed2003

Inhibition of nuclear translocation of transcription factor nuclear factor-kappa B induces FAS- as well as tumour necrosis factor-alpha-mediated apoptosis through downregulation of a conserved family of inhibitor of apoptosis 1.

1. In the present study, we examined whether the nuclear transcription factor (NF)-kappaB activity plays a role in the determination of sensitivity to tumour necrosis factor (TNF)-alpha or agonistic Fas antibody (Ab) in human vascular smooth muscle cells (hVSMC). 2. To inhibit agonist-induced NF-kappaB activation in hVSMC, a cell-permeable peptide (SN50), which carried the nuclear localization sequence of the NF-kappaB p50 subunit, was used. Nuclear factor-kappaB activity was examined by both immunoblot analysis of nuclear extracts and by ELISA. The hVSMC were treated with TNF-alpha or agonistic Fas Ab (CH11) and then apoptosis was determined by cell death ELISA for DNA fragmentation. To investigate the mechanisms for protection against apoptosis in hVSMC, we analysed the expression of a conserved family of inhibitor of apoptosis 1 (c-IAP1) protein using immunoblot analysis. 3. Although both CH11 and TNF-alpha alone failed to induce hVSMC death in the presence of SN50, they markedly increased the apoptotic hVSMC estimated by cell death ELISA. In addition, these effects could be blocked with the pan-caspase inhibitor z-VAD.fmk. Western blotting analysis indicated that TNF-alpha alone increased c-IAP1 protein levels, whereas CH11 alone had no effect. Inhibition of NF-kappaB activation by SN50 suppressed c-IAP1 protein expression and enhanced apoptosis induced by either TNF-alpha or CH11. 4. These findings suggest that c-IAP1 is an important intracellular modulator of Fas as well as TNF-alpha death signalling pathways in hVSMC. The expression of c-IAP1 is regulated by a NF-kappaB-mediated phenomenon.

Active Transport, Cell Nucleus↗
PubMed2003

Oxidized low-density lipoprotein sensitizes human vascular smooth muscle cells to FAS (CD95)-mediated apoptosis.

1. It was investigated in the present study whether oxidized low-density lipoprotein (oxLDL) was implicated in the susceptibility of human vascular smooth muscle cells (VSMC) to Fas-mediated death. Human fetal aorta smooth muscle cells were treated with agonistic anti-Fas antibody (CH11) and oxLDL and cell death was then determined by viability and DNA fragmentation. 2. The results of the present study show that cross-linking of Fas receptor with anti-Fas antibody in the presence of oxLDL induced death and DNA fragmentation in human VSMC, which were blocked by the caspase inhibitor z-VAD.fmk, followed by the upregulation of cell surface Fas. 3. The data indicate that oxLDL is implicated in death in VSMC and provide evidence that oxLDL is involved in Fas signal transduction. The present study proposes a novel mechanism(s) by which VSMC become susceptible to Fas ligand. 4. One of the mechanisms proposed by which oxLDL upregulates cell surface Fas is by inhibiting the degradation of Fas through the ubiquitin-proteasome pathway.

Amino Acid Chloromethyl Ketones↗
PubMed2003

Oxidized low-density lipoprotein inhibits vascular endothelial growth factor-induced endothelial progenitor cell differentiation.

1. Bone marrow-derived endothelial progenitor cells (EPC) in the peripheral blood of adult animals and humans have been shown to be incorporated into neovascularization. In contrast, hypercholesterolaemia impairs angiogenesis and collateral vessel formation in response to regional tissue ischaemia. We investigated whether oxidized LDL (oxLDL) affected human EPC differentiation. 2. When isolated human mononuclear cells (MNC) were incubated with vascular endothelial growth factor (VEGF), the number of differentiated, adherent EPC, as assessed by an in vitro culture assay, was increased in a dose-dependent manner (P < 0.01). When MNC were incubated with oxLDL at 1, 5 and 10 microg/mL in the presence of 100 ng/mL VEGF for 24 h, oxLDL dose-dependently reduced the number of differentiated, adherent EPC. 3. Vascular endothelial growth factor-induced EPC differentiation was significantly inhibited by pharmacological phosphatidylinositol 3-kinase blockers (either 10 nmol/L wortmannin or 10 micromol/L LY294002). Interestingly, immunoblotting analysis revealed that oxLDL dose-dependently led to dephosphorylation and, thus, deactivation of Akt in the presence of VEGF. Finally, these inhibitory effects induced by oxLDL were abolished by pretreatment with 1 micromol/L atorvastatin (P < 0.01). 4. Our data indicate that oxLDL inhibits VEGF-induced EPC differentiation through the dephosphorylation of Akt.

Cell Differentiation↗
PubMed2003

Estrogen attenuates the emotional stress-induced cardiac responses in the animal model of Tako-tsubo (Ampulla) cardiomyopathy.

Reduction of estrogen levels may underlie the high incidence of 'Tako-tsubo (Ampulla) cardiomyopathy in postmenopausal females. Ovariectomized (OVX) and estradiol-supplemented ovariectomized female rats (OVX + E) were subjected to immobilization stress, an animal model of Tako-tsubo cardiomyopathy. In order to evaluate cardiac changes, left ventriculography and electrocardiography were performed under anesthesia (control). Next day, the conscious rats were exposed to immobilization stress, and left ventriculography was performed (stress). In OVX rats, percentage contraction in left ventriculography was significantly reduced in response to stress, while it was not significantly changed in OVX + E rats. In both groups, heart rate was significantly increased in response to stress. However, heart rate in stress was significantly higher in OVX than in OVX + E rats. In summary, these data suggest that increase of serum estradiol levels can diminish the pathological changes in the heart induced by emotional stress.

Animals↗
PubMed2003

Molecular mechanism of emotional stress-induced and catecholamine-induced heart attack.

Emotional or physical stress triggers 'tako-tsubo' cardiomyopathy or 'transient left ventricular apical ballooning', but the pathogenesis is unclear. In response to the immobilization stress of rats, a useful model of emotional stress, rapid activation of p44/p42 mitogen-activated protein kinase was observed in the heart, followed by a transient upregulation of immediate early genes in the smooth muscle cells of coronary arteries, the endothelial cells and the myocardium. Heat shock protein 70 was induced in the aortic and coronary arterial smooth muscle cells and in the myocardium. Natriuretic peptide genes were also upregulated in the myocardium. Sequential gene expression can be considered as an adaptive response to emotional stress. Blocking of both alpha-adrenoceptors and beta-adrenoceptors eliminated the upregulation of immediate early genes induced by stress, while alpha-agonists and beta-agonists upregulated immediate early genes in the perfused heart. Activation of alpha-adrenoceptors and beta-adrenoceptors is the primary trigger of emotional stress-induced molecular changes in the heart.

Animals↗
PubMed2002

Leukemia inhibitory factor relaxes arteries through endothelium-dependent mechanism.

Leukemia inhibitory factor (LIF) is a cytokine, which inhibits angiogenesis and decreases endothelial cell proliferation and migration, suggesting that LIF may modulate vascular tone. In this study, we examined the effects of LIF on the tone of rat arteries. The isometric tension of ring preparations from rat superior mesenteric arteries was continuously measured. LIF relaxed the mesenteric arteries in a dose-dependent manner, when the arterial rings were precontracted with phenylephrine. The relaxation was totally inhibited by mechanical removal of endothelium. N(G)-nitro-L-arginine methyl ester did not affect the relaxation by LIF. Ca(2+)-dependent K channel (KCa) blockers, apamin with charybdotoxin, inhibited the relaxation by LIF. Catalase, an enzyme which scavenges hydrogen peroxide, also inhibited the relaxation by LIF. Endothelium-derived hyperpolarizing factor relaxes smooth muscle cells and the effect is blocked by KCa and catalase. Our results suggest that LIF regulates vascular tone through the effect of this factor.

Animals↗