Cell Biology International (2008) 32, 264-270 - Hai‑Yan Shan and others - Apoptosis is involved in the senescence of endothelial cells induced by angiotensin II

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Cell Biology International (2008) 32, 264–270 (Printed in Great Britain)

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Apoptosis is involved in the senescence of endothelial cells induced by angiotensin II

Hai‑Yan Shan^a, Xiao‑Juan Bai^a^* and Xiang‑Mei Chen^b

^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

Abstract

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.

1 Introduction

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), 2mM l-glutamine, 100U/ml penicillin, and 100μg/ml streptomycin at 37°C in a humidified atmosphere at 5% CO2 in air. Cells were grown to 80–90% confluence in 75cm² culture flasks (Corning, Acton, MA) and were sub-cultured 1:2. Population doublings were calculated as the number of such sub-cultures. For experiments, cells were seeded at 1×10⁴cell/cm² and grown for 48h. This time was chosen to ensure that cells were subconfluent and therefore possible effects of density on growth/response due to contact inhibition were minimal. Subsequently, cells were maintained in serum-free medium for 12h in RPMI-1640 medium to ensure G0 arrest. At this point, Ang II (Sigma, USA) was added into the culture medium and stimulated with a final concentration of 10⁻⁶mol/l for 48h.

2.2 Cell viability assay

To assess the cell viability, 1×10⁴cells/well were seeded in 96-well microtiter plates and allowed to adhere to the plate for 24h. Cells were then treated with 10⁻⁸, 10⁻⁷, 10⁻⁶, 10⁻⁵l or 10⁻⁴mol/l Ang II (each group had 5 wells). Thereafter, 20μl of 5mg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide (MMT, MBI USA) in phosphate-buffered saline (PBS) were added to each well and the cultures were incubated for an additional 4h at 37°C. The supernatant was aspirated and formazan crystals were dissolved in 150μl dimethyl sulfoxide (DMSO). Absorbance at 490nm was read by using an automatic plate reader (Molecular Devices Corp., Sunnyvale, CA). The absorbance values were also called OD values. The cell living percentage was presented as OD experimental cell/OD control cell×100%.

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 10min. They were washed again and incubated with β-galactosidase substrate staining solution (150mM NaCL, 2mM MgCl2, 5mM potassium ferricyanide, 5mM potassium ferrocyanide, 40mM citric acid, and 12mM sodium phosphate, pH 6.0, containing 1mg/ml 5-bromo-4-chloro-3-indolyl-d-β-galactoside) (X-gal) for 24h at 37°C. Senescent cells were identified as blue-stained cells by standard light microscopy, and a minimum of 1000 cells was counted in 10 random fields to determine the percentage of SA-β-gal-positive cells.

2.4 Cell cycle analysis

To analyze cell cycle profiles, cells were digested by 0.25% trypsin containing 1mM EDTA, washed with PBS, and fixed in 70% methanol at 20°C for 10min. Cells were pelleted and resuspended in PBS with RNase A (100μg/ml), incubated for 30min at 37°C, stained with propidium iodide (20μg/ml) for 30min, and analyzed by FACScan flow cytometry (Becton-Dickinson, San Jose, CA) using linear amplification. For each sample, 20,000 events were collected, and clumped cells were gated out. Data were analyzed by an auto-analysis function ModFitLT3.0 software (Verity Software, Topsham, ME).

2.5 Annexin V/PI staining

Cells were incubated with 1μl Annexin V-fluorescein isothiocyanate (BD Pharmingen) in the provided solution and 0.5μl propidium iodide (PI, 10mg/ml; BD Pharmingen) and subsequently analyzed by fluorescence-activated cell sorting (FACScan, Becton-Dickson). PI was added to distinguish between early apoptotic (Annexin V⁺/PI⁻) and late apoptotic or necrotic (Annexin V⁺/PI⁺) cells. Data analysis was performed with Cell Quest software. Cells (10⁴/ml) were counted for each sample.

2.6 Acridine orange fluorescence staining

For morphological identification of apoptosis, cells cultured on 21-cm² dishes were fixed with 4% methanol for 10min at room temperature and stained with 20μg/ml acridine orange solution in PBS for 3min. After staining, cells were washed with water and overlaid with 50% glycerol solution containing 5% 1,4-diazobicyclo-2,2,2-octane as anti-fade. Cells were observed under an inverted fluorescent microscope (TE 300, Nikon, Japan) equipped with a blue excitation filter (B-2A). Nuclei with uneven reddish yellow fluorescent light were classified as apoptotic. A minimum 1000 cells was counted in 10 random fields to determine the percentage of apoptotic cells under fluorescent microscope.

2.7 Transmission electron microscope

Cells were washed twice, and fixed in 2.5% glutaraldehyde in 0.2M cacodylate buffer (pH 7.4) at 4°C for 2h, washed in PBS and post-fixed in 1% OsO4 for 1h. After washing with PBS, the samples were dehydrated in a graded series of ethanol and acetone, and embedded in Epon 812 (Fullen Inc. Latham, NY). Ultrathin sections (70nm) were cut on a Leica ultracut UCT ultramicrotome. After staining with uranyl acetate and lead citrate, sections were observed under Hitachi H500 transmission electron microscope at 75KV.

2.8 Data analysis

All data were expressed as means±standard deviation (SD). Differences were evaluated by t test analysis. Statistical significance was defined as P<0.05.

3 Results

3.1 Cell viability assay

To evaluate the cell viability, HUVEC were treated with various concentrations of Ang II for 48h. MTT assay showed that the viability of cells cultured with 10⁻⁴mol/l and 10⁻⁵mol/l Ang II decreased to 41.2±2.7% and 46.9±3.6%, respectively (P<0.01, n=6) compared to control cells, whereas the viability by 10⁻⁶mol/l Ang II was 81.9±4.1% (P<0.01, n=6). There was no significant difference between the viability of cells cultured with 10⁻⁷mol/l and 10⁻⁸mol/l Ang II compared with control cells. Thus, the senescent model of HUVEC was established by using 10⁻⁶mol/l Ang II.

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±6.81%; P<0.01; see Fig. 1), which indicates that Ang II promoted cellular senescence in HUVEC.

Fig. 1

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 G0 or G1 phase (91.36±6.45%, P<0.01), with few in S and G2/M phases (6.62±0.42% and 2.12±0.33%, P<0.01, respectively; see Fig. 2).

Fig. 2

Cell cycle analysis of the control cells (A, left) and Ang II-induced senescent cells (B, right) by flow cytometry (Red area: G1 phase; Blue area: S phase; Small red area: G2-M phase). The figure shows representative images of 3 independent repetitions.

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±2.4 vs 12.4±2.1%, P<0.01), but their necrosis rates had no significant difference (P>0.05, Fig. 3), which indicated that apoptosis was evident in about 39.6% of cells 48h after exposure to 10⁻⁶mol/l Ang II, concomitantly with expression of senescent phenotype.

Fig. 3

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±2.6 vs 2.8±0.3%, P<0.01; Fig. 4), which proved that apoptosis may participate in the whole process of the senescence of Ang II-stimulated cells.

Fig. 4

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).

Fig. 5

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.

4 Discussion

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 G0/G1 phase, which indicates that Ang II promoted cellular senescence in HUVEC. Jorge and Kurz, 2005 reported that endothelial cells that had undergone replicative senescence in culture show distinctive changes in morphology, such as an increase in size, polymorphic nuclei, flattening and vacuolization, as we have also found. Thus, we have confirmed that Ang II-induced HUVEC senescence and established the senescent model.

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.

Acknowledgments

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 2007

doi:10.1016/j.cellbi.2007.09.003

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