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Cardiovascular Research 2004 61(1):143-151; doi:10.1016/j.cardiores.2003.10.014
© 2004 by European Society of Cardiology
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Copyright © 2004, European Society of Cardiology

Norepinephrine induces apoptosis in neonatal rat endothelial cells via down-regulation of Bcl-2 and activation of β-adrenergic and caspase-2 pathways

Yun-Ching Fua,b, Ching-Shiang Chia,b, Sui-Chu Yinc, Betau Hwangb,d, Yung-Tsung Chiuc and Shih-Lan Hsu*,b,c

aDepartment of Pediatrics, Taichung Veterans General Hospital, Taichung, Taiwan, ROC
bInstitute of Clinical Medicine, National Yang-Ming University, Taipei, Taiwan, ROC
cDepartment of Education and Research, Taichung Veterans General Hospital, Taichung, Taiwan, ROC
dDepartment of Pediatrics, Taipei Veterans General Hospital, Taipei, Taiwan, ROC

* Corresponding author. Department of Education and Research, Taichung Veterans General Hospital, 160, Section 3, Chung-Kang Road, Taichung 40705, Taiwan, ROC. Tel.: +886-4-23592525x4037; fax: +886-4-23592705. h2326{at}vghtc.vghtc.gov.tw

Received 14 July 2003; revised 9 October 2003; accepted 10 October 2003


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusions
 References
 
Objectives: Heart failure is associated with increased plasma norepinephrine (NE) and endothelial apoptosis. Recent reports have suggested that endothelial dysfunction is an important target for future therapies of heart failure. However, whether NE can induce endothelial apoptosis and its mechanism remains unknown. Methods: Endothelial cells from neonatal rat heart were treated with various concentrations of NE for different durations. Apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated nick end-labeling (TUNEL) and DNA fragmentation assays. Caspase activity was measured using specific fluorogenic substrates. Proteins of Bcl-2 family and cytochrome c were assayed by Western blotting. Results: NE induced endothelial apoptosis in a dose- and time-dependent manner. After treatment for 48 h, increasing NE concentration from 5, 10, 50, 100 to 200 µM resulted in 6±3%, 14±5%, 43±4%, 66±5%, and 89±6% apoptotic cells, respectively. The apoptosis was accompanied by down-regulation of Bcl-2 protein synthesis but not by cytosolic cytochrome c translocation. Caspase-2, -3, -6 and -9 were activated during apoptosis and caspase-2 inhibitor (Z-VDVAD-FMK) and caspase-3 inhibitor (Z-DEVD-FMK) significantly attenuated the apoptosis. Overexpression of Bcl-2 inhibited caspase activity and decreased the apoptosis. Moreover, the NE-mediated apoptotic effect was attenuated by β- (β2>β>β1) adrenergic antagonists (ICI-118,551>propranolol>atenolol) but was not affected by {alpha}1- or {alpha}2-adrenergic antagonists (prazosin or yohimbine). Conclusion: Our study is the first report documenting that NE induces apoptosis in neonatal rat endothelial cells mainly through down-regulation of Bcl-2 protein and activation of the β-adrenergic (β2>β1) and caspase-2 pathways. β-Adrenergic antagonists and caspases inhibitors may be useful in the prevention and management of NE-mediated endothelial apoptosis during heart failure.

KEYWORDS Apoptosis; Bcl-2; Caspase; Endothelial cells; Neonatal rat; Norepinephrine


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 5. Conclusions
 References
 
A healthy endothelium plays a central role in cardiovascular control [1]. The endothelial position between tissues and circulating blood assures its simultaneous and constant exposure to a wide variety of stimuli, some of which have the potential to induce apoptosis of endothelial cells [2]. Apoptosis is a form of programmed cell death that results not only in diminished number of cells but also in cellular dysfunction [3]. Endothelial dysfunction participates in the pathogenesis of a number of cardiovascular diseases, including atherosclerosis, hypertension, heart failure, allograft vasculopathy, and reperfusion injury after myocardial ischemia [2,4–7]. Many studies suggest that endothelial dysfunction is an important target for future therapies in patients with heart failure [7–10]. Heart failure is rapidly becoming one of the most prevalent cardiovascular disorders and is characterized by neurohumoral alterations, such as activation of the sympathetic nervous system [11]. Increased plasma norepinephrine (NE) and endothelial apoptosis are observed in patients with heart failure [8–12]. However, the relationship between norepinephrine and endothelial apoptosis has until now not been investigated.

Norepinephrine can induce apoptosis of cardiomyocytes by activation of the β-adrenergic pathway [13]. Zaugg et al. [14] reported that it is selectively mediated by the β1-adrenergic pathway. However, the β2-adrenergic receptor is rare in cardiomyocytes [15] so its role in NE-induced apoptosis needs further clarification. Endothelial cells have abundant {alpha}1 and β2 receptors [16] and become a good model to study different adrenergic pathways involving in NE-induced apoptosis. The molecular mechanisms of induction of apoptosis in different physiological or pathological conditions have been studied intensively [17]. Many molecules including proteins in the Bcl-2 and caspase families participate in the apoptotic response to numerous death-inducing signals [18]. In the present study, we examined the effector mechanisms of NE-induced apoptosis in neonatal rat heart endothelial cells and found that NE-induced endothelial apoptosis was accompanied by down-regulation of Bcl-2 protein synthesis and activation of the β- (β2 >β1) adrenergic and caspase-2 pathways.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 5. Conclusions
 References
 
2.1 Reagents
NE, prazosin, yohimbine, propranolol, atenolol, ICI-118,551, 4',6'-diamidino-2-phenylindole (DAPI), and endothelial cell growth supplement were obtained from Sigma. Caspase-2 inhibitor (Z-VDVAD-FMK), caspase-3 inhibitor (Z-DEVD-FMK), caspase-6 inhibitor (Z-VEID-FMK), caspase-8 inhibitor (Z-IETD-FMK), and caspase-9 inhibitor (Z-LEHD-FMK) were purchased from KAMIYA Biochemical. Antibodies of Bcl-2, Bax, Bcl-XL were obtained from Santa Cruz Biotechnology. Bak, cytochrome c and anti-CD31 (PECAM-1) antibodies were purchased from PharMingen. The recombinant Bcl-2 adenoviral and control adenoviral vectors were kindly provided by Song-Kun Shyue at Institute of Biomedical Sciences, Academia Sinica.

2.2 Isolation of neonatal rat endothelial cells
Endothelial cells were prepared from 4-day-old neonatal Sprague–Dawley rat hearts as described elsewhere [19]. Briefly, the neonatal rat hearts were removed from donors, washed with Hanks balanced salt solution (HBSS) and finely minced with dissecting scissors. The minced tissue was subjected to four successive trypsinization steps (0.125% trypsin in 10 ml calcium- and magnesium-free HBSS, at 37 °C for 6 min) under stirring in a 50-ml flask. After trypsinization, free cells were collected by centrifugation at 600 x g, then resuspended in Dulbecco's modified essential medium (DMEM) containing 15% fetal bovine serum, and allowed to adhere to the fibronectin-coated plates. After 90-min incubation, the culture plates were washed with HBSS to remove nonadherent cells (mostly cardiomyocytes). The remaining adherent cells (endothelial cells) were maintained in DMEM containing 15% fetal bovine serum, 50 µg/ml endothelial cell growth supplement, 100 IU/ml penicillin and 100 µg/ml streptomycin. The growth medium was changed every 2 days. Endothelial cell cultures were identified by indirect anti-desmin (1:200; Sigma) and anti-Von Willibrand factor (1:200; Santa Cruz Biotechnology) immunofluorescence stain [20]. Further identified the isolated endothelial cells by flow cytometry using mouse anti-rat CD31 (PECAM-1) antibody. The obtained endothelial cell cultures were more than 99% pure without significant contamination of other types of cells (Fig. 1). This project was approved by the Institutional Animal Care and Use Committees of the Taichung Veterans General Hospital. The investigation conforms by the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).


Figure 1
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  		Fig. 1 Flow cytometric analysis of CD31 (PECAM-1) expression: cultured rat endothelial cells and cardiomyocytes were harvested with 5 mM EDTA and stained for surface expression of CD31 (PECAM-1) using a PE-conjugated mouse anti-rat CD31 antibody, and expression was analyzed by flow cytometry. Histograms are composites with the expression of CD31 on cells in black, negative controls in white.

 
2.3 Flow cytometric analysis of CD31 (PECAM-1) expression
Cultured rat endothelial cells and cardiomyocytes cells were washed with PBS and detached using 5 mM EDTA. Cells were suspended in 1% bovine serum albumin in PBS for 30 min at 4 °C. After incubation, a phycoerythrin (PE)-conjugated mouse anti-CD31 antibody was added for 2 h at 4 °C, then washed three times with PBS. Analysis of CD31 surface expression was performed on a flow cytometer (Becton Dickinson Instruments). Mouse IgG1 conjugated with PE was used as a negative control staining (Fig. 1).

2.4 Cell treatments and apoptosis assay
Endothelial cells in serum-free medium were treated with various concentrations of NE for different durations, or were pretreated with different adrenergic antagonists for 30 min before the addition of NE for 48 h. The treatments included NE alone (100 µM), NE+prazosin ({alpha}1-adrenergic antagonist, 1 µM), NE+yohimbine ({alpha}2-adrenergic antagonist, 5 µM), NE+propranolol (β-adrenergic antagonist, 1 µM), NE+atenolol (β1-adrenergic antagonist, 5 µM), and NE+ICI-118,551 (β2-adrenergic antagonist, 1 µM). The apoptosis was determined using in situ terminal deoxynucleotidyl transferase-mediated nick end-labeling (TUNEL) assay (Roche Diagnostics, Meylan, France). Then cells were washed and double stained with 1 µg/ml DAPI at room temperature for another 20 min. The labeled endothelial cells were examined by fluorescence microscopy. To quantify apoptotic cells, the percentage of TUNEL positive cells (with green fluorescent nuclei) was measured at 200 x magnification in five randomly chosen fields of each nine replicates from three independent cultures. The proportion of TUNEL-positive cells was expressed as a percentage of the total cells counted. Some endothelial cells were transduced with recombinant Bcl-2-adenoviral and control adenoviral vectors for 16 h prior to exposure to NE. The cells were treated for 2 h with caspase inhibitors prior to exposure to NE, apoptotic cells were estimated.

2.5 DNA fragmentation analysis
Rat heart endothelial cells were treated with or without 10, 50 and 100 µM NE for 48 h. Total cells (both attached and floating cells) were harvested and resuspended in 400 µl of ice-cold lysis buffer (containing 50 mM Tris–HCl, pH 7.5, 10 mM EDTA, 0.3% Triton X-100), incubated on ice for 30 min, and then centrifuged. The supernatant was incubated with Proteinase K (200 µg/ml) for 2 h, at 50 °C. RNaseA (100 µg/ml) was added and incubated at 50 °C for another 2 h. Fragmented DNA was extracted with phenol/chloroform, and precipitated with ethanol/sodium acetate at –20 °C. The DNA fragments were electrophoresed on a 2% agarose gel containing 0.1 µg/ml ethidium bromide.

2.6 Measurement of caspase activity
Caspase-2, -3, -6, -8, and -9 activities were determined 12, 24 and 48 h after treatment of endothelial cells with NE (100 µM). Briefly, endothelial cells were lysed to release their intracellular contents. The cell lysate was then tested for caspase activity by addition of a caspase-specific peptide substrate conjugated with the fluorescent reporter molecule 7-amino-4-trifluoromethyl coumarin (R&D System Minneapolis, USA). The cleavage of the peptide by the caspase releases the fluorochrome that when excited by light at 400 nm emits fluorescence at 505 nm. The level of caspase enzymatic activity in the cell lysate is directly proportional to the fluorescence signal detected with a fluorescent microplate reader (Fluoroskan Ascent; Labsystems, Finland).

2.7 Protein preparation and western blot analysis
Apoptotic and anti-apoptotic proteins (Bak, Bax and Bcl-2, Bcl-XL) were assayed by Western blot. Briefly, endothelial cells were lysed in ice-cold buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 10 µg/ml soybean trypsin inhibitor, 0.5 mM phenylmethylsulfonyl fluoride) for 30 min at 4 °C. The supernatant was collected and protein concentrations were determined by the Bradford method. Equal amounts of total protein were loaded onto SDS-polyacrylamide gels and the proteins electrophoretically transferred to a PVDF membrane (Millipore, Bedford, MA). Immunoblots were analyzed using specific primary antibodies against Bcl-2, Bcl-XL, Bak, Bax, and β-actin that were diluted 500-, 500-, 1000-, 500-, and 2000-fold, respectively. After exposure to horseradish peroxidase-conjugated secondary antibody for 1 h, proteins were visualized using an enhanced chemiluminescence detection kit (Amersham, Aylesbury, UK). To evaluate mitochondrial cytochrome c release, fractions enriched in mitochondria and cytosol were prepared [21,22]. Briefly, endothelial cells were collected by centrifugation at 600 x g for 10 min. The cell pellets were washed twice with ice-cold phosphate-buffered saline and resuspended in homogenization buffer containing 20 mM Tris–HCl (pH 7.4), 250 mM sucrose, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 10 µg/ml aprotinin. The cells were homogenized in a glass Dounce homogenizer with tight pestle for 30 strokes at 4 °C. The homogenates were centrifuged at 900 x g for 10 min at 4 °C to remove unbroken cells and nuclei. The supernatants were then centrifuged at 10,000 x g for 15 min at 4 °C, and the resulting mitochondrial pellets were resuspended in homogenization buffer. Mitochondria were further purified on percol gradient according to the manufacturer's recommendations (Amersham Biosciences). Cytosolic fraction was achieved by centrifuging the supernatant at 100,000 x g for 1 h at 4 °C. Protein concentrations were determined and samples were frozen as aliquots at –80 °C for subsequent experiments. The fractions were analyzed by probing Western blots with anti-tubulin antibody (BD Transduction Laboratories) to track cytosolic fraction, and anti-OxPhos complex IV antibody (Molecular Probes) to track mitochondrial fraction.

2.8 Statistical analysis
All data are expressed as mean±S.D. obtained from nine independent experiments with similar patterns. Student's unpaired t-test was used to compare groups. The mean values of two groups were considered significantly different if p<0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 5. Conclusions
 References
 
3.1 NE-induced apoptosis in rat heart endothelial cells
To determine whether NE induces apoptotic cell death, neonatal rat heart endothelial cells were exposed to NE for 48 h, TUNEL assay was performed. Under a fluorescence microscopy, many green-fluorescent cells (TUNEL-positive cells) were observed after 48 h NE (100 µM) treatment (Fig. 2A). Fewer or no TUNEL-positive cells were detected in control cultures. In addition, treatment with NE resulted in degradation of chromosomal DNA into small internucleosomal fragments, as evidenced by the formation of a 180–200 bp DNA ladder on the agarose gels (Fig. 2B). These observations suggest that NE triggered apoptotic cell death in endothelial cells. Moreover, the NE-induced apoptosis was dose-dependent. As shown in Fig. 3A, 1 µM NE treatment failed to induce apoptosis; whereas by increasing NE concentration from 5, 10, 50, 100 to 200 µM, approximately 6±3%, 14±5%, 43±4%, 66±5%, and 89±6% apoptotic cells were detected, respectively. In control cultures less than 1%, apoptotic cells were observed. To examine the kinetics of apoptotic death induced by NE treatment, we performed a time course analysis. The results in Fig. 3B indicate that NE (50 and 100 µM) caused time-dependent cell death, approximately 15±3%, 39±5%, and 70±6% apoptotic cells were observed for 12-, 24-, and 48-h incubation with 100 µM NE.


Figure 2
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  		Fig. 2 Induction of endothelial apoptosis by NE. Endothelial cells were untreated or treated with 100 µM NE for 48 h. (A) TUNEL assay and DAPI staining were performed. Scale bar, 50 µm. (B) DNA fragmentation analysis. Cells were treated with or without NE (10, 50 and 100 µM) for 48 h. After treatment, DNA was isolated and electrophoresed on a 2% agarose gel.

 

Figure 3
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  		Fig. 3 NE induced endothelial apoptosis in a dose- and time-dependent manner. (A) Dose dependence. Endothelial cells were incubated with various concentrations of NE (1 to 200 µM) for 48 h. (B) Time-course dependence. Endothelial cells were incubated with 50 and 100 µM NE for 0, 12, 24, and 48 h. After treatment, apoptotic death was estimated by TUNEL assay. The percentage of TUNEL-positive endothelial cells of each treatment is presented as the mean±S.D. of nine independent experiments.

 
3.2 Activation of caspases involved in NE-mediated apoptosis
Activation of the caspase cascade is a central mechanism of apoptosis, and caspases are believed to be the executioners in cell death [18]. To elucidate whether caspase family proteases are activated in the NE-induced apoptotic process, the proteolytic activity of specific caspase was measured using fluorogenic peptide substrates. Fig. 4A shows that caspase-2 was the first activated caspase during NE-induced endothelial apoptosis. The activities of caspase-2, -3, -6 and -9 were significantly increased 48 h after NE treatment. However, caspase-8 was not activated by NE. To define whether activation of a particular caspase is required for induction of apoptosis by NE, specific caspase inhibitors were used. The results show that the NE-induced apoptotic cell death was significantly blocked by treatment with caspase-2 inhibitor (Z-VDVAD-FMK) and caspase-3 inhibitor (Z-DEVD-FMK), but not by caspase-6 inhibitor (Z-VEID-FMK), caspase-8 inhibitor (Z-IETD-FMK) or caspase-9 inhibitor (Z-LEHD-FMK) (Fig. 4B). At 100 µM, the caspase-2 and -3 inhibitors restored cell viability by 75% and 80%, respectively (Fig. 4B). These observations indicate that caspase-2 and -3 are integrally involved in NE-triggered endothelial apoptosis. To further examine the role of caspase-2 in NE-induced apoptosis, the activity of caspase-2, -3, -6 and -9 in the cell lysates after NE treatment in the presence of the caspase-2 inhibitor was examined. As depicted in Fig. 4C, Z-VDVAD-FMK markedly blocked the activation of all four caspases in endothelial cells treated with 100 µM NE for 48 h. These observations suggest that activation of caspase-2 occurs before the activation of caspase-3, -6 and -9 in NE-mediated endothelial cell apoptosis.


Figure 4
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  		Fig. 4 Involvement of caspases in NE-induced apoptosis. (A) Activation of caspases. Endothelial cells were treated with 100 µM NE for 12, 24, and 48 h. After treatment, cell lysate was prepared, and caspase activities of caspase-2, -3, -6, -8 and -9 were measured using specific fluorogenic substrates. (B) Prevention of NE-induced apoptosis by caspase inhibitors. Endothelial cells were treated with or without 100 µM NE for 48 h following pretreatment for 2 h in the presence or absence of various concentrations of caspase inhibitors (Z-VDVAD-FMK, Z-DEVD-FMK, Z-VEID-FMK, Z-IETD-FMK and Z-LEHD-FMK). After treatment, apoptotic cell was measured by TUNEL assay. *p<0.05, compared with the NE-treated group. (C) Blockade of activation of caspase-2, -3, -6 and -9 by a caspase-2 inhibitor. Cells were treated with 100 µM NE and/or 100 µM caspase-2 inhibitor (Z-VDVAD-FMK) for 48 h. Activity of caspase-2, -3, -6, -8 and -9 in cell lysates was measured by using their specific fluorogenic substrates. *p<0.05 vs. NE treatment alone.

 
3.3 Down-regulation of Bcl-2 protein during NE-induced apoptosis
The Bcl-2 family of proteins plays an important role in the regulation of the apoptotic pathway [23]. To determine whether Bcl-2-related molecules are modulated in NE-triggered endothelial apoptosis, we examined the expression levels of several Bcl-2 family members by Western blot analysis. As illustrated in Fig. 5, exposure of endothelial cells to 100 µM NE resulted in a significant decrease in Bcl-2 protein levels compared to controls. However, the levels of Bak, Bax and Bcl-XL proteins were unaffected by NE treatment.


Figure 5
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  		Fig. 5 Decrease of Bcl-2 protein level in NE-treated cells. Endothelial cells were treated with or without 100 µM NE for 12, 24, and 48 h. After treatment, total protein was extracted. The expressions of Bcl-2, Bcl-XL, Bak and Bax proteins were analyzed by Western blot. β-actin was used as an internal loading control.

 
3.4 Attenuation of NE-induced apoptosis and caspase activation by Bcl-2 overexpression
To address the effect of Bcl-2 in NE-induced apoptosis, endothelial cells were transduced with 50 multiplicity of infection (moi) of Bcl-2-adenoviral and control adenoviral vectors. As indicated in Fig. 6A, the expression level of Bcl-2 protein was increased by Bcl-2 adenoviral vector infection compared to that of control adenoviral infection. Moreover, overexpression of Bcl-2 protein by adenoviral infection drastically prevented NE-triggered apoptosis (Fig. 6A) and reduced the activity of caspase-2, -3, -6 and -9 (Fig. 6B). These results indicate that Bcl-2 acts upstream of the caspase cascade.


Figure 6
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  		Fig. 6 Attenuation of NE-induced apoptosis by Bcl-2 overexpression. Endothelial cells were transduced with Bcl-2-adenoviral and control adenoviral vectors for 16 h, and then treated with or without 100 µM NE for another 48 h. After treatment, (A) the expression of Bcl-2 protein and apoptotic cell number were determined; and (B) the activity of caspase-2, -3, -6, -8 and -9 was detected. *p<0.05, compared with the NE-treated group.

 
3.5 NE-triggered apoptosis did not involve cytochrome c release
Bcl-2 protein is a mitochondrial membrane protein which can alter the permeability of the mitochondrial membrane and trigger the release of cytochrome c, caspase-2 and caspase-9, then activate post-mitochondrial caspases, including caspase-3, -6 and -7 [23]. To determine whether cytochrome c release contributes to the proapoptotic effects of NE, we observed whether its subcellular localization was changed by NE treatment. As depicted in Fig. 7, no translocation of cytochrome c from the mitochondria to the cytosol could be detected at the time points analyzed (12, 24 and 48 h), suggesting cytochrome c release is not involved in the NE-induced apoptotic pathway.


Figure 7
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  		Fig. 7 The effects of NE on mitochondrial cytochrome c release. Endothelial cells were treated with or without 100 µM NE for indicated time points. After incubation, mitochondrial and cytosolic fractions were isolated and analyzed by Western blot analysis using cytochrome c specific antibody.

 
3.6 Effect of adrenergic antagonists on NE-induced endothelial apoptosis
Previous reports have demonstrated that NE stimulates apoptosis in rat heart myocytes by activation of the β-adrenergic pathway [13,14]. To test the possibility that NE elicits its apoptotic effect on endothelial cells via stimulation of adrenergic receptor pathway, the antagonist of {alpha}1- (prazosin), {alpha}2- (yohimbine), β- (propranolol), β1- (atenolol), and β2- (ICI-118,551) receptors were used in follow-up experiments. Results showed that administration of endothelial cells with the antagonist of adrenergic receptors alone did not affect cell viability (data not shown). However, the β-adrenergic antagonists significantly attenuated NE-induced apoptotic cell death, following a gradient of ICI-118,551>propranolol>atenolol (Fig. 8). In contrast, {alpha}1- or {alpha}2-adrenergic antagonist treatment did not affect the NE-induced apoptosis (Fig. 8).


Figure 8
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  		Fig. 8 Effect of antagonist of adrenergic receptors on NE-induced apoptosis in endothelial cells. The endothelial cells were treated with 100 µM NE alone, or NE combined with prazosin (Pra, {alpha}1 antagonist), yohimbine (Yoh, {alpha}2 antagonist), propranolol (Pro, β antagonist), atenolol (Ate, β1 antagonist), or ICI-118,551 (ICI, β2 antagonist). Apoptotic cells were determined by TUNEL assay after 48 h treatment. Data depicted are means of nine independent experiments. *p<0.05, compared with the NE-treated group.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
5. Conclusions
 References
 
The sympathetic nervous system has been viewed as the physiologically critical mechanism for eliciting cardiovascular response to increased circulatory needs during acute stress, augmenting cardiac rate and contractility and changing peripheral vascular tone [24]. However, excessive and prolonged activity of this system plays a pivotal role in the pathogenesis of cardiovascular diseases [11]. Heart failure is characterized by prolonged activation of the sympathetic nervous system and is associated with impaired endothelial function [7,11]. It has been postulated that exposure to high levels of catecholamines is toxic to cardiac myocytes and endothelial cells. Romeo et al. [25] demonstrated that epinephrine can induce apoptosis in human coronary artery endothelial cells. NE is the primary transmitter of the sympathetic nervous system [26]. However, whether NE is able to induce apoptosis of endothelial cells has not been investigated before. Defining the mechanisms through which NE contributes to endothelial death could help to identify potential treatment strategies to prevent heart failure. Our present study provides biochemical evidence that the reduction of Bcl-2 protein levels and activation of the caspase-2 cascade and the β-adrenergic pathway constitute early events of the NE-induced apoptotic pathway and it is not accompanied by subcellular translocation of cytochrome c.

Despite the lack of cytochrome c release from mitochondria, multiple caspases (including caspase-2, -3, -6, and -9) are activated in NE-induced endothelial apoptosis. Our observations showed that caspase-2 like protease was activated earlier and more dramatically than other caspases. Moreover, NE-induced apoptosis and caspase activation were markedly inhibited by a caspase-2 inhibitor (Z-VDVAD-FMK), suggesting a crucial role of the caspase-2 like protease in the initiation of apoptosis induced by NE in endothelial cells. Caspase-2 is considered to be both an initiator and effector caspase [18,27–29]. Previous studies have indicated that caspase-2 is directly cleaved by the effector caspase-3, suggesting a possible downstream involvement of this caspase in the apoptotic cascade [30,31]. However, caspase-2 also has characteristics of an initiator caspase acting upstream of the effector caspases [32–35]. Our finding that inhibition of caspase-2 prevented cell death and abolished caspase-3, -6 and -9 activities in NE-treated endothelial cells implicates caspase-2 like protease acting as a regulatory caspase upstream of caspase-3, -6 and -9. These results suggest that caspase-2 activation may represent another major class of apoptotic pathways distinct from that mediated by caspase-8 and caspase-9.

The anti-apoptotic protein Bcl-2 plays an important role in controlling cell death [23]. We observed that NE-treated endothelial cells decreased the Bcl-2 protein level, whereas Bax, Bcl-XL, and Bak levels were unchanged. Overexpression of Bcl-2 in NE-treated endothelial cells was capable of restoring cell viability and also abrogated caspase activities, suggesting that Bcl-2 acts upstream of the caspase activation. These results are in agreement with previous reports showing that Bcl-2 acts upstream of the caspase cascade [36–40].

In the vascular system, NE exerts its effect by binding to adrenergic receptors, which belong to the large multigenic family of receptors coupled to GTP-binding proteins [41]. Several subtypes of adrenergic receptors have been identified: {alpha}1, {alpha}2, β1, β2, and β3. Accumulating evidence has indicated that the effect of NE depends on the quantitative distribution of different subtype receptors. Zaugg et al. [14] reported that NE-induced apoptosis in cardiomyocytes is selectively mediated by the β1-adrenergic pathway. Because the major adrenergic receptors in cardiomyocytes are β1 receptors [15], it is not surprising that the effect of NE on cardiomyocytes is mediated by the β1 adrenergic pathway. However, the roles of {alpha} and β2 receptors in NE-induced apoptosis need further clarification. It has been reported that adrenergic receptors {alpha}1 and β2 are dominantly expressed in endothelial cells [16]. Here we found that NE can induce endothelial apoptosis by activation of the β- (β2>β1) but not {alpha}-adrenergic pathway. The β-blockers only partially inhibited NE-induced apoptosis, suggesting β-adrenergic receptor pathway may not be the only pathway, and involves other pathway(s) in NE-triggered endothelial cell apoptosis. It is documented that β receptors (β1 or β2) can activate the cAMP-protein kinase A pathway via activation of adenylyl cyclase. Several in vitro studies have demonstrated that activation of the cAMP-protein kinase A pathway is involved in the NE-triggered apoptotic process [13,42]. This is the first report documenting that NE induces apoptosis in neonatal rat endothelial cells by activation of the β-adrenergic pathway. Whether the cAMP-protein kinase A pathway is necessary for NE-induced apoptosis in endothelial cells and the detailed downstream signaling remains to be explored.

At this stage, how does NE regulate Bcl-2 protein expression and activate the caspase-2 cascade? The possible relationship between the activation of β-adrenergic receptors and the activation of caspases or down-regulation of Bcl-2 during NE-triggered apoptotic death remain unclear and are the subject of ongoing research in our laboratory.

The epidemics of enterovirus 71 infections caused the rapid death of many children in Malaysia in 1997 and in Taiwan in 1998 [43]. Most of the deceased patients are infants. All patients experienced sympathetic hyperactivity including hypertension, tachycardia etc., prior to the onset of acute heart failure and pulmonary edema. Our previous study postulated the mechanism as below [43]: enterovirus 71 encephalitis, mainly in the brain stem, results in a catecholamine storm, which damages cardiovascular system and causes acute heart failure and pulmonary edema. Based on the present study, β-adrenergic antagonists may be able to prevent the heart failure and pulmonary edema in patients with enterovirus 71 encephalitis. However, in order to be relevant for adult pathology, further researches investigating adult rat or human endothelial cells are needed.

In conclusion, the present study provides biochemical evidence that apoptotic events caused by NE in neonatal rat heart endothelial cells occurred mainly via the pathway involving Bcl-2 and caspases but not cytochrome c release. Moreover, NE-triggered apoptosis occurs at least in part by the activation of the β-adrenergic pathway. β2-Adrenergic antagonist can more significantly attenuate the apoptosis than β1-adrenergic antagonist. These observations suggest that β-adrenergic antagonists and caspase inhibitors may be potent agents to be used in the prevention and management of endothelial apoptosis caused by sympathetic system over-activation.


   5. Conclusions
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusions
References
 
Our study is the first report documenting that NE induces apoptosis in neonatal rat endothelial cells mainly through down-regulation of Bcl-2 protein and activation of β-adrenergic (β2>β1) and caspase-2 pathways. β-Adrenergic antagonists and caspases inhibitors may be useful in the prevention and management of NE-mediated endothelial apoptosis during heart failure.


   Acknowledgements
 
This study was supported by a grant from Cardiac Children's Foundation, Taiwan.


   Notes
 
Time for primary review 29 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusions
 References
 

  1. Cines D.B., Pollak E.S., Buck C.A., et al. Endothelial cells in physiology and in the pathophysiology of vascular disorders. Blood (1998) 91:3527–3561.[Free Full Text]
  2. Stefanec T. Endothelial apoptosis: could it have a role in the pathogenesis and treatment of disease? Chest (2000) 117:841–854.[CrossRef][Web of Science][Medline]
  3. Thompson C.B. Apoptosis in the pathogenesis and treatment of disease. Science (1995) 267:1456–1462.[Abstract/Free Full Text]
  4. Poredos P. Endothelial dysfunction in the pathogenesis of atherosclerosis. Int. Angiol. (2002) 21:109–116.[Web of Science][Medline]
  5. Ferrari R., Bachetti T., Agnoletti L., Comini L., Curello S. Endothelial function and dysfunction in heart failure. Eur. Heart J. (1998) 19(Suppl. G):G41–G47.[Web of Science][Medline]
  6. Lefer A.M., Tsao P.S., Lefer D.J., Ma X.L. Role of endothelial dysfunction in the pathogenesis of reperfusion injury after myocardial ischemia. FASEB J. (1991) 5:2029–2034.[Abstract]
  7. Lopez Farre A., Casado S. Heart failure, redox alterations, and endothelial dysfunction. Hypertension (2001) 38:1400–1405.[Abstract/Free Full Text]
  8. Katz S.D. Mechanisms and implications of endothelial dysfunction in congestive heart failure. Curr. Opin. Cardiol. (1997) 12:259–264.[Web of Science][Medline]
  9. Sharma R., Davidoff M.N. Oxidative stress and endothelial dysfunction in heart failure. Congest. Heart Fail. (2002) 8:165–172.[CrossRef][Medline]
  10. Rossig L., Haendeler J., Mallat Z., et al. Congestive heart failure induces endothelial cell apoptosis: protective role of carvedilol. J. Am. Coll. Cardiol. (2000) 36:2081–2089.[Abstract/Free Full Text]
  11. Tavazzi L., Opasich C. Clinical epidemiology of heart failure. Congest. Heart Fail. (1999) 5:260–269.[Medline]
  12. Pepper G.S., Lee R.W. Sympathetic activation in heart failure and its treatment with beta-blockade. Arch. Intern. Med. (1999) 159:225–234.[Abstract/Free Full Text]
  13. Communal C., Singh K., Pimentel D.R., Colucci W.S. Norepinephrine stimulates apoptosis in adult rat ventricular myocytes by activation of the beta-adrenergic pathway. Circulation (1998) 98:1329–1334.[Abstract/Free Full Text]
  14. Zaugg M., Xu W., Lucchinetti E., Shafiq S.A., Jamali N.Z., Siddiqui M.A. Beta-adrenergic receptor subtypes differentially affect apoptosis in adult rat ventricular myocytes. Circulation (2000) 102:344–350.[Abstract/Free Full Text]
  15. Stiles G.L., Taylor S., Lefkowitz R.J. Human cardiac beta-adrenergic receptors: subtype heterogeneity delineated by direct radioligand binding. Life Sci. (1983) 33:467–473.[CrossRef][Web of Science][Medline]
  16. Howell R.E., Albelda S.M., Daise M.L., Levine E.M. Characterization of beta-adrenergic receptors in cultured human and bovine endothelial cells. J. Appl. Physiol. (1988) 65:1251–1257.[Abstract/Free Full Text]
  17. Vaux D.L., Korsmeyer S.J. Cell death in development. Cell (1999) 96:245–254.[CrossRef][Web of Science][Medline]
  18. Hengart M.O. The biochemistry of apoptosis. Nature (2000) 407:770–776.[CrossRef][Medline]
  19. Richards P.S., Saba T.M., Del Vecchio P.J., Vincent P.A., Gray V.C. Matrix fibronectin disruption in association with altered endothelial cell adhesion induced by activated polymorphonuclear leukocytes. Exp. Mol. Pathol. (1986) 45:1–21.[CrossRef][Web of Science][Medline]
  20. Antin P.B., Mar J.H., Ordahl C.P. Single cell analysis of transfected gene expression in primary heart cultures containing multiple cell types. BioTechniques (1988) 6:640–649.[Web of Science][Medline]
  21. Yang S.E., Hsieh M.T., Tsai T.H., Hsu S.L. Down-modulation of Bcl-XL, release of cytochrome c and sequential activation of caspases during honokiol-induced apoptosis in human squamous lung cancer CH27 cells. Biochem. Pharmacol. (2002) 63:1641–1651.[CrossRef][Web of Science][Medline]
  22. Rosse T., Olivier R., Monney L., et al. Bcl-2 prolongs cell survival after bax-induced release of cytochrome c. Nature (1998) 391:496–499.[CrossRef][Medline]
  23. Adams J.M., Cory S. Life-or-death decisions by the Bcl-2 protein family. Trends Biochem. Sci. (2001) 26:61–66.[CrossRef][Web of Science][Medline]
  24. Cohn J.N. Sympathetic nervous system activity and the heart. Am. J. Hypertens. (1989) 2:353S–356S.[Medline]
  25. Romeo F., Li D., Shi M., Mehta J.L. Carvedilol prevents epinephrine-induced apoptosis in human coronary artery endothelial cells: modulation of Fas/Fas ligand and caspase-3 pathway. Cardiovasc. Res. (2000) 45:788–794.[Abstract/Free Full Text]
  26. Udenfriend S. Biosynthesis of the sympathetic neurotransmitter, norepinephrine. Harvey Lect. (1966) 60:57–83.[Medline]
  27. Rabkin S.W. Prevention of staurosporine-induced cell death in embryonic chick cardiomyocyte is more dependent on caspase-2 than caspase-3 inhibition and is independent of sphingomyelinase activation and ceramide generation. Arch. Biochem. Biophys. (2001) 390:119–127.[CrossRef][Web of Science][Medline]
  28. Lassus P., Opitz-Araya X., Lazebnik Y. Requirement for caspase-2 in stress-induced apoptosis before mitochondrial permeabilization. Science (2002) 297:1352–1354.[Abstract/Free Full Text]
  29. Guo Y., Srinivasula S.M., Druilhe A., Fernandes-Alnemri T., Alnemri E.S. Caspase-2 induces apoptosis by releasing proapoptotic proteins from mitochondria. J. Biol. Chem. (2002) 277:13430–13437.[Abstract/Free Full Text]
  30. Mancini M., Machamer C.E., Roy S., Nicholson D.W., Thornberry N.A., Casciola-Rosen L.A., et al. Caspase-2 is localized at the golgi complex and cleaves golgin-160 during apoptosis. J. Cell Biol. (2000) 149:603–612.[Abstract/Free Full Text]
  31. Slee E.A., Adrain C., Martin S.J. Serial killers: ordering caspase activation events in apoptosis. Cell Death Differ. (1999) 6:1067–1074.[CrossRef][Web of Science][Medline]
  32. Ahmad M., Srinivasula S.M., Wang L., et al. CRADD, a novel human apoptotic adaptor molecule for caspase-2, and FasL/tumor necrosis factor receptor-interacting protein RIP. Cancer Res. (1997) 57:615–619.[Abstract/Free Full Text]
  33. Duan H., Dixit V.M. RAIDD is a new "death" adaptor molecule. Nature (1997) 385:86–89.[CrossRef][Medline]
  34. Huo H., Luo R., Metz S.A., Li G. Activation of caspase-2 mediates the apoptosis induced by GTP-depletion in insulin-secreting (HIT-T15) cells. Endocrinology (2002) 143:1695–1704.[Abstract/Free Full Text]
  35. Chen W., Wang H.G., Srinivasula S.M., Alnemri E.S., Cooper N.R. B cell apoptosis triggered by antigen receptor ligation proceeds via a novel caspase-dependent pathway. J. Immunol. (1999) 163:2483–2491.[Abstract/Free Full Text]
  36. Boulakia C.A., Chen G., Ng F.W., et al. Bcl-2 and adenovirus E1B19 kDa protein prevent E1A-induced processing of CPP32 and cleavage of poly(ADP-ribose) polymerase. Oncogene (1996) 12:529–535.[Web of Science][Medline]
  37. Orth K., Chinnaiyan A.M., Garg M., Froelich C.J., Dixit V.M. The CED-3/ICE-like protease Mch2 is activated during apoptosis and cleaves the death substrate lamin A. J. Biol. Chem. (1996) 271:16443–16446.[Abstract/Free Full Text]
  38. Srinivasan A., Foster L.M., Testa M.P., et al. Bcl-2 expression in neural cells blocks activation of ICE/CED-3 family proteases during apoptosis. J. Neurosci. (1996) 16:5654–5660.[Abstract/Free Full Text]
  39. Haviv R., Lindenboim L., Yuan J.Y., Stein R. Need for caspase-2 in apoptosis of growth-factor-deprived PC12 cells. J. Neurosci. Res. (1998) 52:491–497.[CrossRef][Web of Science][Medline]
  40. Heinke M.Y., Yao M., Chang D., Einstein R., dos Remedios C.G. Apoptosis of ventricular and atrial myocytes from pacing-induced canine heart failure. Cardiovasc. Res. (2001) 49:127–134.[Abstract/Free Full Text]
  41. Strosberg A.D. Structure, function, and regulation of adrenergic receptors. Protein Sci. (1993) 2:1198–1209.[Web of Science][Medline]
  42. Iwai-Kanai E., Hasegawa K., Araki M., Kakita T., Morimoto T., Sasayama S. Alpha- and beta-adrenergic pathways differentially regulate cell type-specific apoptosis in rat cardiac myocytes. Circulation (1999) 100:305–311.[Abstract/Free Full Text]
  43. Fu Y.C., Chi C.S., Jan S.L., et al. Pulmonary edema of enterovirus 71 encephalomyelitis is associated with left ventricular failure: implications for treatment. Pediatr. Pulmonol. (2003) 35:263–268.[CrossRef][Web of Science][Medline]

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