Takuzo Hano

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 β-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.

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 β-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.

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.

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 β-galactosidase staining. 17β-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. 17β-Estradiol significantly increased telomerase activity. Interestingly, reverse transcriptase-PCR analysis demonstrated that 17β-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 17β-estradiol on Akt activity in EPCs. Immunoblotting analysis revealed that 17β-estradiol dose-dependently led to phosphorylation and, thus, to activation of Akt in EPCs. We also examined whether the protective effect of 17β-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 17β-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 17β-estradiol. Finally, in a Matrigel assay, EPCs treated with both 17β-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.

Catecholamines and estrogen are involved in the pathogenesis of emotional stress-induced acute heart attack.

Emotional stress triggers takotsubo cardiomyopathy in postmenopausal women. Clinical analysis of autonomic nervous function has revealed a transient increase of sympathetic nervous activity and decrease of vagal nervous activity. Immobilization (IMO) stress of rats can reproduce the electrocardiographic and left ventriculographic changes that occur in takotsubo cardiomyopathy, both of which are prevented by combined blockade of α‐ and β‐adrenoceptors. Estrogen supplementation partially attenuated these cardiac changes. It also attenuated the IMO‐induced increase of c‐Fos immunoreactivity, or c‐fos mRNA expression in the lateral septum, medial amygdaloid nucleus, paraventricular hypothalamic nucleus, dorsomedial hypothalamic nucleus, laterodorsal tegmental nucleus, and locus ceruleus; these regions contain central sympathetic neurons and neurons with immunoreactive estrogen receptors. It also downregulated c‐fos mRNA expression in the adrenal gland and the heart, suggesting an increase of estrogen attenuated the stress‐induced hypothalamo‐sympathoadrenal outflow from the central nervous system to the target organs. Estrogen treatment also upregulated the levels of cardioprotective substances, such as atrial natriuretic peptide and heat shock protein 70, in the heart. These data suggest that reduction of estrogen levels following menopause might be involved in the primary cause of takotsubo cardiomyopathy both by indirect action on the nervous system and by direct action on the heart.

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.

Cellular FLIP is expressed in cardiomyocytes and down-regulated in TUNEL-positive grafted cardiac tissues

OBJECTIVE c-FLIP is a natural homologue of caspase 8, and may antagonize activation of death pathways mediated by FADD. c-FLIP is highly expressed in the heart, and a recent report suggests that c-FLIP may protect against certain types of myocyte death. The present study was designed to define the expression patterns of c-FLIP in the heart. METHODS The expression pattern of c-FLIP in end-stage human hearts, and rat cardiomyocyte grafting models was analyzed by in situ hybridization, immunohistochemistry and TUNEL assay. In addition, to determine whether Fas-dependent pathway is active in cardiomyocytes in vitro, we examined whether activated monocytes can kill neonatal cardiomyocytes in a co-culture system. RESULTS c-FLIP mRNA and protein were abundantly expressed in normal cardiomyocytes from failing human heart. In animal models, c-FLIP protein was absent in TUNEL-positive grafted cardiomyocytes. Double staining demonstrated that c-FLIP-positive cells rarely had fragmented DNA, while TUNEL-positive cells rarely contained c-FLIP. Finally, activated monocytes induced death of neonatal rat cardiomyocytes via the Fas/FasL system. CONCLUSIONS Loss of c-FLIP expression correlates with cardiomyocyte cell death. We hypothesize that diminished c-FLIP expression may predispose cardiomyocytes to apoptotic death.

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 17β-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 β-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.

Apoptosis of vascular smooth muscle cells is induced by Fas ligand derived from monocytes/macrophage

Abstract Fas and its ligand (FasL), are a receptor-ligand pair identified as promoting cell death in several tissues. Apoptosis of vascular smooth muscle cells (VSMCs) in human atherosclerotic plaque may contribute to weakening of the fibrous cap, ultimately resulting in plaque rupture. We investigated the ability of monocytes to induce apoptosis of cultured VSMCs through Fas/FasL pathway. In addition, we examined the association of FasL with apoptosis in human coronary plaques. Both activated monocytes and the supernatant obtained from activated monocytes were able to kill cultured VSMCs. The apoptotic response of VSMCs was almost completely blocked by the caspase inhibitor z-VAD-fmk and was partially blocked by incubation with antagonistic anti-Fas IgG1 which suggests that Fas/FasL system was involved in the induction of cell death. An ≈30 kDa protein, which represents a cleaved, soluble form of FasL, was identified in culture medium from activated monocytes, but not in culture medium from control, unactivated monocytes. Immunohistochemical analysis of human atherosclerotic coronary lesions showed that FasL is expressed by macrophages, and microvessels in the adventitia as well as in the plaque. Finally, double-staining with terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) and FasL antibody showed that FasL enriched lesions always included a number of TUNEL-positive cells. These data suggest that Fas/FasL pathway can be employed by monocytes/macrophages to induce VSMC apoptosis in the atherosclerotic lesions.

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. 17β-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 β-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.