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Cell Biology International (2008) 32, 264270 (Printed in Great Britain)
Apoptosis is involved in the senescence of endothelial cells induced by angiotensin II
Hai‑Yan Shana, Xiao‑Juan Baia* and Xiang‑Mei Chenb
aDepartment of Cardiology, The First Affiliated Hospital of China Medical University, No.155, Nanjing North street, Heping District, Shenyang, Liaoning Province 110001, China
bDepartment of Nephrology, General Hospital of People's Liberation Army, Beijing 100853, China
Vascular endothelial cells have a finite cell lifespan and eventually enter an irreversible growth arrest, cellular senescence. The functional changes associated with cellular senescence are thought to contribute to human aging and age-related cardiovascular disorders, e.g. atherosclerosis. In this study, induction of Angiotensin II (Ang II) promoted a growth arrest with phenotypic characteristics of cell senescence, such as enlarged cell shapes, increased senescence-associated β-galactosidase (SA-β-gal) positive staining cell, and depressed cell proliferation. Apoptotic changes were increased in senescent cells, with a small subset of the senescent cells showing aberrant morphology such as pronounced nuclear fragmentation or multiple micronuclei. The results suggest cell apoptosis is possibly an important factor in the process of pathologic and physiologic senescence of endothelial cells as well as vascular aging.
Keywords: Angiotensin II, Senescence, Apoptosis, Atherosclerosis, Endothelial cell, Ultrastructure.
*Corresponding author. Tel.: +86 024 8328 2687; fax: +86 024 8328 2693.
Cardiovascular disease is the most common cause of death among the elderly, which is associated with aging and affects cardiovascular function, e.g., atherosclerosis (Docherty, 1990; Folkow and Svanborg, 1993; Lakatta, 1993; Dohi et al., 1995; Kuniya et al., 2000; Shipley and Muller, 2005). The incidence of atherosclerosis increases with age. Aging is associated with endothelial dysfunction, a key pathogenic factor in atherosclerotic disease progression (Zeiher et al., 1993; Schachinger et al., 2000). On a cellular level, advancing age impairs endothelial function (Hoffmann et al., 2001). Evidence of endothelial dysfunction and biochemical patterns resemble early atherosclerosis (Asai et al., 2000; Csiszar et al., 2002; Ferrari et al., 2003).
The endothelium is located in strategic anatomical position within the blood vessel wall and thereby acts as a barrier between blood and vascular smooth muscle cells. Therefore, the functional integrity of the endothelium monolayer is essential to prevent vascular leakage and the formation of atherosclerotic lesion. The senescence endothelial cells may critically disturb the integrity of the endothelial monolayer and may thereby contribute to vascular injury and atherosclerosis (Ross et al., 1984; Copper et al., 1994; Hoffmann et al., 2001; Minamino et al., 2002).
Angiotensin II (Ang II), the primary effector of the renin-angiotensin system (RAS), is a multifunctional hormone that plays a major role in regulating blood pressure and cardiovascular homeostasis. Recent evidences suggest that Ang II may also play an important role in aging. For example, senescence correlates with increased synthesis of cardiac Ang II and decreased synthesis of plasma Ang II (Heymes et al., 1998a). Moreover, the densities of Ang II receptor type I (AT1) and receptor type II (AT2) are increased in myocardium of senescent rats (Heymes et al., 1998b), and up-regulation of AT1 may be involved in initiation and progression of atherosclerosis (Chen et al., 2002), and endothelial dysfunction is associated with aging in rats (Kansui et al., 2002; Mukai et al., 2002). It is assumed that Ang II promotes vascular cell senescence, thereby contributing to the pathogenesis of human atherosclerosis.
Based on above information, the purpose of this study was to test the hypothesis that Ang II may induce HUVEC senescence. In addition, because cell apoptosis could be the mechanism for vascular dysfunction and atherosclerosis in the elder, we further explored the role of cell apoptosis in the aging process, where the ultrastructure changes and apoptosis incidence of endothelial cells were observed.
2 Materials and methods
2.1 Cell culture
Human umbilical vein endothelial cells (HUVEC) (American Type Culture Collection, USA) were cultured in RPMI-1640 medium (GIBCO-BRL, Gaithersburg, MD) supplemented with 10% (v/v) FBS (Hyclone, Logan, UT), 2
2.2 Cell viability assay
To assess the cell viability, 1
2.3 Senescence-associated β-galactosidase (SA-β-gal) staining
The senescent status of cells was verified by in situ staining for SA-β-galactosidase (see Dimri et al., 1995). Briefly, cells growing on 21-cm2 cell culture dishes were washed with PBS for 3 times, and fixed with 2% formaldehyde/2% glutaraldehyde in PBS for 10
2.4 Cell cycle analysis
To analyze cell cycle profiles, cells were digested by 0.25% trypsin containing 1
2.5 Annexin V/PI staining
Cells were incubated with 1
2.6 Acridine orange fluorescence staining
For morphological identification of apoptosis, cells cultured on 21-cm2 dishes were fixed with 4% methanol for 10
2.7 Transmission electron microscope
Cells were washed twice, and fixed in 2.5% glutaraldehyde in 0.2
2.8 Data analysis
All data were expressed as means
3.1 Cell viability assay
To evaluate the cell viability, HUVEC were treated with various concentrations of Ang II for 48
3.2 SA-β-gal staining in senescent HUVEC
Cellular senescence in vascular endothelial cells is induced in atherosclerotic plaque. We first determined whether Ang II induced cellular senescence in HUVEC. We performed SA-β-gal staining, a reliable biomarker for cellular senescence. Increased staining of SA-β-gal (blue color) has been reported in aging cells when assayed at close neutral pH (Dimri et al., 1995; Kurz et al., 2000). In the present study, SA-β-gal-positive endothelial cells appeared flattened and enlarged in contrast to the round shape of control cells. The percentage of SA-β-gal staining was significantly increased in Ang II-induced cells compared to that of control cells (80.10
Senescence-associated-β-galactosidase (SA-β-Gal) staining in HUVEC. Images are of the control cells (A, left) and Ang II-induced senescent cells (B, right). Original magnification was ×400. The figure represents a representative experiment of 3 independent repetitions.
3.3 Cell cycle analysis
Flow cytometry showed that cells were mostly in G
Cell cycle analysis of the control cells (A, left) and Ang II-induced senescent cells (B, right) by flow cytometry (Red area: G
3.4 Annexin V/PI staining in senescent HUVEC
Although apoptosis in vivo has been shown to be involved in the aging process, in vitro studies of age-dependent apoptosis are limited. In our study, apoptosis was examined in HUVEC exposed to Ang II to induce premature senescence. Early stage apoptosis was followed through phosphatidylserine (PS) in the lining of the endothelium which was exposed in the inside-out orientation of the cell membrane. Annexin V labeled with fluorescein isothiocyanate (FITC) has an especially high chemical affinity for PS, which may detect the early period apoptosis. We examined Annexin V-FITC/PI double-staining by flow cytometry. The early-metaphase apoptotic rate was significantly increased in Ang II-induced cells compared to that of control cells (39.6
HUVEC stained with acridine orange. (A) The control cells; (B) Ang II-induced senescent cells showing slight enlargement and fragmented nuclei. Original magnification was ×400. The figure represents a typical experiment of 3 independent repetitions.
3.5 Acridine orange fluorescence staining in senescent HUVEC
Cells undergoing apoptosis could be morphologically identified by uneven reddish yellow fluorescent in cytoplasm and nuclei by using acridine orange fluorescence staining. The morphological changes of apoptosis in HUVEC were observed under a confocal microscope (Leica, Germany). The percentage of apoptotic cells in Ang II-stimulated cells was significantly increased compared to that of the control cells (31.8
The early stage apoptosis was detected by flow cytometry with Annexin V-FITC/PI double staining. Flow cytometry graphs of the control cells (A, left) and Ang II-induced senescent cells (B, right). The figure represents a typical experiment of 3 independent repetitions.
3.6 Ultrastructure observations
In the transmission electron microscopy, control cells appeared round and smooth in shape and had even chromatin (Fig. 5A), but senescent cells appeared flattened and enlarged. Cells with chromatin condensing at the nuclear margin, showing invaginations of nuclear membrane, and cytoplasmic vacuolarization were classified as aging (Fig. 5B). The apoptotic morphology in senescent HUVEC included irregular shapes in the nuclei, heterochromatin condensation along nuclear membranes (Fig. 5C), nuclear fragmentation and apoptotic bodies (Fig. 5D).
Ultrastructure of HUVEC by transmission electron microscope. Photograph of the control cell (A). Original magnification was ×8000. Ang II-induced senescent cell (B–D), Original magnification was ×6000. The figure represents typical examples from 3 independent experimental repetitions.
Since senescent cells acquire characteristics that may compromise normal tissue function, their accumulation in later life has been postulated to contribute to the aging process and to the development of age-related diseases, such as atherosclerosis (Campisi, 2005). The accumulation of senescent cells in the arterial wall may contribute to both initiation and progression of atherosclerosis (Ross, 1993; Minamino et al., 2003; Brandes et al., 2005). Thus, it is important to study the biologic properties of senescent cells.
Ang II, the primary effector of the renin-angiotensin system (RAS), is a multifunctional hormone that plays a major role in regulating blood pressure and cardiovascular homeostasis. Ang II may also play an important role in aging. Recent studies suggest that Ang II stimulation of EPC increases gp91phox expression, which may contribute to oxidative stress, as evidenced by peroxynitrite formation, and therefore Ang II induced EPC senescence via increased oxidative stress (Imanishi et al., 2004, 2005a,b). Therefore, in our study, we tested the hypothesis that Ang II may induce HUVEC senescence.
Cellular senescence is a response phenomenon resulting in a permanent withdrawal from the cell cycle and the appearance of distinct morphological and functional changes associated with an impairment of cellular homeostasis (Jorge and Kurz, 2005). As in most other mammalian cells, the division capacity of endothelial cells is limited and ultimately the cells enter a state of irreversible growth arrested senescence (Foreman and Tang, 2003). Senescence cells are metabolically active but morphologically altered and express senescence-associated enzymes such as the acidic β-galactosidase (SA-β-gal). An increased SA-β-gal activity has been observed in endothelial cells within atherosclerotic plaques (Minamino et al., 2002). In this study, we observed that SA-β-gal staining was significantly increased in Ang II-stimulated cells compared to that of the control cells, cell cycle analysis showed that the cells arrested in the G
In addition, cell senescence is an aging-associated or a vascular disease-associated phenomenon. Senescent endothelial cells are more prone to proapoptotic stimuli (Matsushita et al., 2001). Passaging of endothelial cells per se renders them susceptible to apoptosis (Hoffmann et al., 2001). Litter doubt that endothelial cell apoptosis can occur in vivo. Various stimuli, such as inflammatory cytokines, Ang II, oxidized lipids and turbulent blood flow seem to promote this process (Dimmeler et al., 2002). Here, we also observed that the early-metaphase apoptotic rate was significantly increased by using Annexin V-FITC/PI double staining, the morphological changes of apoptosis in HUVEC was observed under a confocal microscope and transmission electron microscopy, the apoptotic cells remarkably increased in Ang II-stimulated cells compared to the control cells, which proved that apoptosis may participate in the whole process in the senescence of Ang II-stimulated cells.
A potential mechanism for the endothelial senescence was the increased density of apoptotic cells observed in the Ang II-stimulated cells compared with the control cells. Although the initial cause of increased apoptosis in aging endothelium is not clear, it may be due to endothelial cell aging through apoptosis. It is well known that senescence in vascular endothelial cells initiates atherosclerosis (Minamino et al., 2004). However, apoptosis of endothelial cells is essential for the initiation of atherosclerosis, has not been previously reported. The results obtained in this study demonstrate that a small subset of HUVEC undergoing apoptosis concomitant with cellular senescence.
Although much evidence indicates the apoptosis pathway is activated in aging tissues or cells, the role of apoptosis in the aging process is poorly understood. It is also possible that the senescent phenotype induced by Ang II in this study does represent authentic senescence. If the latter is the case, it is important that its validity should be confirmed. Further studies on mechanisms leading to age-associated apoptosis involved in vitro senescent cells may contribute to our understanding of the role of apoptosis in the aging process.
We thank Dr Chang Ying (Institut Pasteur de Lille, France) for discussion and reading of the manuscript, we are also grateful to Dr Lei Yang (China Medical University) and Dr Si-yang Zhang (China Medical University) for valuable technical contribution. The study was supported by a grant from Major Basic Project of China (973), No. G2007CB507405.
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Received 9 May 2007/31 July 2007; accepted 4 September 2007doi:10.1016/j.cellbi.2007.09.003