Cardiovascular Research

Cardiovascular Research 1999 41(1):109-115; doi:10.1016/S0008-6363(98)00196-5
© 1999 by European Society of Cardiology

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Copyright © 1999, European Society of Cardiology

Modulation of constitutive nitric oxide synthase, bcl-2 and Fas expression in cultured human coronary endothelial cells exposed to anoxia–reoxygenation and angiotensin II: role of AT1 receptor activation

Dayuan Li, Kathleen Tomson, Baichun Yang, Paulette Mehta, Byron P. Croker and Jawahar L. Mehta^*

Departments of Medicine, Physiology, Pediatrics and Pathology, University of Florida and VA Medical Center, 1600 Archer Rd., P.O. Box 100277, JHMHC Gainesville, FL 32610, USA

^* Corresponding author. Tel.: +1-(352)-379-4160; fax: +1-(352)-379-4161; e-mail: mehta@medmac.ufl.edu

Received 17 February 1998; accepted 4 May 1998

	Abstract

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Background: Angiotensin II (Ang II) plays a critical role in the pathophysiology of myocardial ischemia–reperfusion injury. We have recently shown that reoxygenation following a period of anoxia causes apoptosis of cultured human coronary artery endothelial cells (HCAECs). Ang II further enhances apoptosis of HCAECs via Ang II type 1 receptor (AT1R) activation. Recent studies suggest an important role of constitutive nitric oxide synthase (cNOS), Fas and bcl-2 proteins in apoptosis. This study was designed to examine the modulation of cNOS, and Fas and bcl-2 expression in HCAECs during exposure to anoxia–reoxygenation and Ang II. Methods and Results: HCAECs were exposed to anoxia–reoxygenation and Ang II. Anoxia- reoxygenation significantly decreased cNOS mRNA, protein and activity in cultured HCAECs (P<0.05 vs. control). Anoxia–reoxygenation also caused an increase in Fas and a decrease in bcl-2 protein expression in cultured HCAECs (both P<0.05 vs. control). The presence of Ang II (0.3 µM) further enhanced these effects of anoxia–reoxygenation on cNOS, Fas and bcl-2 expression. The effects of Ang II were significantly attenuated by the AT1R inhibitor losartan (10 µM). Conclusion: During exposure of HCAECs to anoxia–reoxygenation and Ang II, AT1R activation induces important changes in cNOS mRNA, protein expression and activity, as well as bcl-2 and Fas protein expression which may have a bearing on the development of apoptosis.

KEYWORDS Ang II, Angiotensin II; AT1R, Angiotensin II type 1 receptor; AT2R, Angiotensin II type 2 receptor; cNOS, Constitutive nitric oxide synthase; HCAECs, Human coronary artery endothelial cells; iNOS, Inducible nitric oxide synthase

	1 Introduction

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Angiotensin II (Ang II), formed as a result of the activation of renin–angiotensin system, participates in the pathogenesis of myocardial ischemia–reperfusion injury [1]. The critical role of renin–angiotensin system is evident from significant cardioprotective effects of angiotensin-converting enzyme inhibitors in animals as well as in humans [2, 3]. The cardiovascular effects of Ang II are initiated when Ang II interacts with at least two pharmacologically distinct subtypes of cell-surface receptors, Ang II type 1 and type 2 [4]. While the activation of Ang II type 1 receptor (AT1R) causes vasoconstriction and exacerbates myocardial ischemia–reperfusion injury [1], activation of Ang II type 2 receptor (AT2R) inhibits proliferation of endothelial cells [5].

Nitric oxide (NO) appears to play diverse roles in different tissues. At least two different isoenzymes of NO synthase (NOS) exist: one is a constitutive calcium-dependent isoform (cNOS), and the other is cytokine-inducible and calcium-independent isoform (iNOS). The ability of vascular endothelial cells and smooth muscle cells to express both cNOS and iNOS suggests that different isoforms contribute to the control of vascular tone in physiologic and pathologic states. For example, a recent study showed that inhibition of cNOS exaggerated myocardial dysfunction, whereas inhibition of iNOS improved myocardial function [6]. Several studies have suggested that Ang II and NO are involved in apoptosis (programmed cell death). For example, Pollman et al. [7]showed that Ang II can directly antagonize NO donor- and cGMP analogue-induced apoptosis via activation of AT1R. Accordingly, modulation of Ang II and NO synthesis and activity may have important theoretical and clinical implications in cardiovascular disease.

Reoxygenation following a period of anoxia activates apoptosis in rabbit cardiomyocytes [8]as well as endothelial cells [9]. Although the precise trigger of apoptosis during ischemia reperfusion is unknown, it is evident that expression of bcl-2 protein prevents apoptosis [10], whereas activation of Fas gene induces apoptosis in response to diverse stimuli [11, 12]. In recent studies from our laboratory, we have shown that Ang II enhances apoptosis in cultured human coronary artery endothelial cells (HCAECs) during anoxia–reoxygenation. The pro-apoptotic effects of Ang II appear to involve protein tyrosine kinase and protein kinase C signal conduction pathways as well as release of free oxygen radicals [9]. We also observed that these effects of Ang II were significantly attenuated by losartan, a specific AT1R blocker, indicating that it is the AT1R activation which mediates the pro-apoptotic effects of Ang II on HCAECs.

In the present study, we examined modulation of cNOS, bcl-2 and Fas in HCAECs exposed to anoxia–reoxygenation and Ang II.

	2 Methods

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
2.1 Cell culture
HCAECs were purchased from Clonetics Corporation (Lot no. 6F0756). Microvascular endothelium growth medium consisted of 500 ml of endothelial cell basal medium, 5 ng human recombinant epidermal growth factor, 0.5 mg hydrocortisone, 25 mg gentamycin and 25 µg amphotericin B, 6 bovine brain extract and 25 ml fetal bovine serum. HCAECs in 5 ml MV-EGM were seeded in a 25 cm² flask (4000 cells/cm²), incubated at 37°C in air–CO₂ (95:5). Fourth subcultivation HCAECs (1·10⁶) were used in these experiments, as described earlier [9]. The cells were examined under phase-contrast microscopy, and when about 85% confluent, culture medium was changed, and the cells were divided into four groups:

Control group: cells were incubated in air–CO₂ (95:5) for 27 h

Anoxia–reoxygenation group: cells were exposed to anoxia (N₂–CO₂, 95:5) for 24 h followed by reoxygenation (air–CO₂, 95:5) for 3 h

Ang II plus anoxia–reoxygenation group: cells were incubated with Ang II (0.3 µM) and then exposed to anoxia–reoxygenation condition. The concentration of human sequence Ang II (Sigma) used in the present study was chosen on the basis of previously published literature [13]

Losartan plus Ang II plus anoxia–reoxygenation group: cells were incubated with the specific AT1R blocker losartan (10 µM) and Ang II and then exposed to anoxia–reoxygenation. The dose of losartan used in the present study was chosen on the basis of IC₅₀ of losartan and previous work [14].

2.2 Determination of cNOS mRNA, protein and activity in cultured HCAECs
2.2.1 mRNA expression
Reverse transcriptase polymerase chain reaction (RT-PCR) was performed for identification of NOS mRNA in these experiments. Extraction of total RNA in different groups of cultured HCAECs was based on the single step method of Chomczynski and Sacchi [15]. RNA was subjected to reverse transcription by incubating with oligo dT, four dNTPs, RNAse inhibitor, moloney leukemia virus reverse transcriptase (M-LV-RT), dithiothreitol (DTT) and RT buffer (Gibco-BRL).

cNOS was amplified using the following primers: 5'-CAGTGTCCAACATGCTGCTGGA AATTG-3' and 5'-TAAAGGTCTTCTTCCTGGTGATGCC-3', bases 1050–1076 (sense) and 1511–1535 (antisense), respectively, amplifying a 485 base pair product [16]. cNOS was amplified in the presence of primers, MgCl₂ (1 mM), dNTPs, buffer and Taq DNA polymerase (Promega) and cycled in a Perkin-Elmer 480 DNA Thermo Cycler (96°C x 35 s, 62°C x 2 min, and 72°C x 2 min) for 40 cycles. The RT-PCR amplified samples were visualized on 1.5% agarose gels using ethidium bromide. ß-Actin was amplified as a reference for quantitation of cNOS.

2.2.2 Protein expression
Cells were solubilized directly in boiling 2x concentrated electrophoresis sample buffer (1x=125 mM Tris–HCl pH 6.8, 2% SDS, 12.5 µg/ml aprotinin, 5 µM leupeptin, 5% glycerol, 0.003% bromophenol blue, and 1% ß-mercaptoethanol) and centrifuged at 12 500 g for 15 min at 4°C. The cytosolic protein from different groups of cells (10 µg per lane) was separated by 8% SDS-PAGE by use of a Bio-Rad (Richmond, CA, USA) mini-protein cell, transferred to nitrocellulose filters (Amersham Life Science), and then immunoblotted with a mouse monoclonal antibody against human cNOS (Transduction Laboratories) at 1:250 dilution. Anti-human alkaline phosphatase-conjugated antibody was used as a secondary antibody at 1:4000 dilution. Mouse monoclonal antibody against rat neuronal NOS was used as a negative control. Human endothelial cell lysate (Transduction Laboratories) was used as a positive control. The blot was directly detected for color development on membrane. Sites of antigen localization turn a dark purple color as a result of alkaline phosphatase activity. Total protein content of different samples was quantified by BCA^® protein assay kit (Pierce). Relative intensities of bands of interest were analyzed by use of an MSF-300G Scanner (Microtek Lab) [17].

2.2.3 NOS activity
For measurement of cNOS activity, cells were incubated with ³H-L-arginine (10 µl, average count 2000 000 cpm) for 1 h at 37°C in NO buffer (25 mM Hepes, 140 mM NaCl, 5.4 mM KCl, 1 mM CaCl₂, 1 mM MgCl₂, 0.09% glucose, pH 7.4). The reaction was terminated with 1 ml of stop buffer (118 mM NaCl, 4.7 mM KCl, 1.18 mM KH₂PO₄, 24.8 mM NaHCO₃, 4 mM EDTA, 5 mM N-nitro-L-arginine methylester, pH 5.5). Cells were washed twice and lysed by addition of 1 ml of 0.3 M HClO₄. A 65-µl volume of 3 M K₂CO₃ was then added to neutralize the HClO₄. A 500-µl volume of the mixture was removed for scintillation spectroscopy for determination of total count. Another 500 µl of mixture was applied to Dowex AG50WX-8 (Na⁺ form) columns (Bio-Rad) and eluted with 2 ml of distilled water. Conversion of ³H-L-arginine to ³H-L-citrulline was determined as (Dowex eluent/total count)x100%. Details of the methodology and its validation have been described by us elsewhere [17].

2.3 Determination of bcl-2 and Fas protein expression
Cells were solubilized directly in boiling 2x concentrated electrophoresis sample buffer and centrifuged to obtain cytosol as described above. The cytosolic protein from aliquots of different groups of cells (10 µg per lane) was separated by 8% SDS-PAGE by use of a Bio-Rad mini-protein cell, transferred to nitrocellulose filters (Amersham), and then immunoblotted with a rabbit monoclonal antibody against human bcl-2 (Transduction Laboratories) at 1:800 dilution. Anti-human alkaline phosphatase conjugated antibody was used as a secondary antibody at 1:4000 dilution. The process of determination of Fas protein was similar to the procedure used for bcl-2 analysis, except for the use of rabbit monoclonal antibody against human Fas [18].

2.4 Data analysis
For the purpose of calculation of data, control values for cNOS mRNA and cNOS, Fas and bcl-2 protein were expressed as one arbitrary unit (A.U.). All data represent mean of duplicate samples from at least five independently performed experiments. Data are presented as mean±S.D. Multiple comparisons among independent groups of data were made by ANOVA and the F test. A P value of 0.05 was considered statistically significant.

	3 Results

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
3.1 Determination of cNOS mRNA, protein and activity
3.1.1 cNOS mRNA by RT-PCR
Anoxia–reoxygenation decreased cNOS mRNA expression (cNOS band density: 0.50±0.05 vs. 1.00 A.U. in control group, P<0.05, n=5). The presence of Ang II further decreased cNOS mRNA expression (cNOS band density: 0.08±0.01 A.U., P<0.05 vs. anoxia–reoxygenation alone, n=5). The effect of Ang II was inhibited by losartan (cNOS band density: 0.34±0.03 A.U., P<0.05 vs. Ang II+anoxia–reoxygenation, n=5). A representative experiment along with summary of data from five separate experiments are shown in Fig. 1.

View larger version (62K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Amplification of cNOS mRNA by RT-PCR in HCAECs. Groups of HCAECs were exposed to anoxia–reoxygenation (A–R), Ang II+A–R, or losartan+Ang II+A–R before preparation of RNA for amplification. Note that A–R decreases cNOS mRNA, and Ang II further enhances the decrease in cNOS mRNA beyond that caused by A–R. The effect of Ang II is blocked by the AT1R blocker losartan. See text for details of the methodology. Upper panel shows results of one experiment and the lower panel shows densitometric analysis of five separate experiments. Bars are values in mean±S.D.

 
ß-Actin expression, as index of amount of amplified mRNA in each group, was similar among four groups.

3.1.2 cNOS protein expression
Anoxia–reoxygenation decreased cNOS protein expression (cNOS band density: 0.78±0.05 vs. 1.00 A.U. in control group, P<0.05, n=5). The presence of Ang II further decreased cNOS protein expression (band density: 0.63±0.07 A.U., P<0.05 vs. anoxia–reoxygenation alone, n=5), and this effect was inhibited by losartan (band density: 0.85±0.06 A.U., P<0.05 vs. Ang II+anoxia–reoxygenation, n=5) (Fig. 2).

View larger version (51K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Identification of cNOS protein in HCAECs by Western analysis. Groups of HCAECs were exposed to anoxia–reoxygenation (A–R), Ang II+A–R, or losartan+Ang II+A–R before preparation of protein for separation. Note that A–R decreases cNOS protein, and Ang II enhances the effect of A–R. The effects of Ang II are attenuated by losartan. See text for details of the methodology. Upper panel shows results of one experiment and the lower panel shows densitometric analysis of five separate experiments. Bars are values in mean±S.D.

 
3.1.3 cNOS activity
In accordance with the data on cNOS mRNA and protein expression, anoxia–reoxygenation decreased cNOS activity compared with the control group (P<0.05, n=6). The presence of Ang II further decreased cNOS activity compared with anoxia–reoxygenation alone (P<0.01, n=6), and this effect was attenuated by losartan compared with Ang II+anoxia–reoxygenation (P<0.05, n=6) (Fig. 3).

View larger version (19K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Determination of cNOS activity in HCAECs by conversion of 3H-L-arginine to 3H-L-citrulline. Groups of HCAECs were exposed to anoxia–reoxygenation (A–R), Ang II+A–R, or losartan+Ang II+A–R before incubation with 3H-L-arginine. Note that A–R decreases cNOS activity, and Ang II enhances the effect of A–R. The effects of Ang II are attenuated by losartan. See text for details of the methodology. Bars represent values in mean±S.D.

 
3.2 Determination of bcl-2 and Fas protein expression
Anoxia–reoxygenation decreased bcl-2 protein expression (bcl-2 band density: 0.67±0.06 vs. 1.00 A.U. in the control group, P<0.05, n=5). The presence of Ang II further decreased bcl-2 protein expression (bcl-2 band density: 0.55±0.14 A.U., P<0.05 vs. anoxia–reoxygenation alone, n=5), and this effect was inhibited by losartan (bcl-2 band density: 0.85±0.12 A.U., P<0.05 vs. Ang II+anoxia–reoxygenation, n=5) (Fig. 4).

View larger version (31K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 Identification of Fas and Bcl-2 protein in HCAECs by Western analysis. Groups of HCAECs were exposed to anoxia–reoxygenation (A–R), Ang II+A–R, or losartan+Ang II+A–R before preparation of protein for separation. Note that A–R decreases Bcl-2 protein and increases Fas protein, and Ang II enhances these effects of A–R. The effects of Ang II are attenuated by losartan. See text for details of the methodology. These results are representative of five separate experiments.

 
Anoxia–reoxygenation increased Fas protein expression (Fas band density: 1.14±0.17 vs. 1.00 A.U. in the control group, P<0.05, n=5). The presence of Ang II further increased Fas protein expression (Fas band density: 1.40±0.14 A.U., P<0.05 vs. anoxia–reoxygenation alone, n=5), and this effect was inhibited by losartan (Fas band density: 0.96±0.13 A.U., P<0.05 vs. Ang II+anoxia–reoxygenation, n=5) (Fig. 4).

	4 Discussion

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
This study shows that anoxia–reoxygenation decreases cNOS mRNA, protein and activity in cultured HCAECs. Ang II further enhances the effects of anoxia–reoxygenation on cNOS. Anoxia–reoxygenation also increases Fas and decreases bcl-2 protein expression in cultured HCAECs. The presence of Ang II further enhances the effects of anoxia–reoxygenation on Fas and bcl-2 protein expression. Most importantly, these effects of Ang II can be blocked by the AT1R blocker losartan, indicating that the effects of Ang II are mediated by AT1R activation.

4.1 Anoxia–reoxygenation, Ang II and modulation of cNOS in cultured HCAECs
The most effective therapy of myocardial ischemia is restoration of coronary blood flow. However, reperfusion per se may lead to additional tissue injury by induction of apoptosis and necrosis [8, 9]. Activation of L-arginine–NO and renin–angiotensin systems may have a critical role in this process. Reperfusion of previously ischemic myocardial tissues results in endothelial dysfunction, increase in coronary vascular resistance and extension of myocardial injury [19]. This so-called ‘reperfusion injury’ has been attributed to the loss of release of NO and/or its inactivation by release of superoxide anions [20]. In accordance with this hypothesis, augmentation of synthesis, release or activity of NO has been shown to be beneficial in some models of ischemia–reperfusion [21, 22].

In a previous study from our laboratory, we observed that anoxia–reoxygenation causes apoptosis of HCAECs, and Ang II via AT1R activation augments the effect of anoxia–reoxygenation. We also observed that anoxia–reoxygenation causes lipid peroxidation, indicating release of free radicals; Ang II further increases lipid peroxidation via AT1R activation because losartan markedly reduces Ang II-mediated lipid peroxidation [9]. The present study was designed to address the modulation of cNOS mRNA, protein and activity which may play an important role in determining injury to the cultured HCAECs following anoxia–reoxygenation. We indeed found that 3 h of reoxygenation following 24 h of anoxia decreases cNOS mRNA, protein and activity. These observations suggest that anoxia–reoxygenation may affect the transcription of cNOS. We believe that the decrease in cNOS activity plays an important role in anoxia–reoxygenation-mediated injury to HCAECs. Liao et al. [23]showed that a decrease in O₂ concentration from 95 to 3% produces a progressive decrease in cNOS mRNA and protein levels in bovine pulmonary artery endothelial cells. A decrease in O₂ concentration from 95 to 3% also shortened the half life of cNOS mRNA and caused a 20-fold suppression of cNOS gene transcription. Inhibition of cNOS has been shown to cause vasoconstriction, a decline in cardiac output and increased mortality, as shown in endotoxemic rats [24]and in rabbits with myocardial infarction [6]. Similar harmful effects of inhibition of cNOS on blood pressure and myocardial infarction have been reported by others as well [21, 25].

We also found that the presence of Ang II decreased cNOS mRNA, protein expression and activity during anoxia–reoxygenation, and these effects were inhibited by the AT1R blocker, losartan, indicating that the effects of Ang II on the transcription of cNOS are mediated at least in part via AT1R activation. We believe that the remainder of the decrease in cNOS mRNA, protein expression and activity is caused by release of free radicals and in part by endothelial cell death during anoxia–reoxygenation. Importantly, anoxia–reoxygenation per se does not alter AT1R mRNA expression (determined by RT-PCR, authors’ unpublished data). We [9]and others [26]have shown that functional responses to Ang II in endothelial cells are predominantly mediated by AT1R activation. However, in contrast to the reduction in cNOS activity in HCAECs exposed to anoxia, Saito et al. [26]reported a rapid (within 1 min) and dose-dependent (10^–9–10^–6 M) increase in cNOS activity in response to Ang II in bovine endothelial cells, which was also mediated via AT1R activation. Thus the effects of Ang II on cNOS may well be time-dependent. It is noteworthy that NO has been shown to be a bifunctional modulator of cell fate capable of either stimulating or inhibiting cell death, depending on the cell type [27].

4.2 Anoxia–reoxygenation, Ang II and modulation of Fas and bcl-2 expression in cultured HCAECs
Although molecular pathways controlling apoptosis in cardiac cells have not been precisely defined, it is known that bcl-2 expression inhibits apoptosis and Fas expression promotes this process. Bcl-2 protein [10]has been shown to prevent apoptosis induced by diverse stimuli by acting as an antioxidant or by mechanisms unrelated to its effect on reactive oxygen radicals. Studies have shown that downregulation of bcl-2 expression promotes apoptosis in human umbilical vein endothelial cells [28]. Enforced expression of bcl-2 gene using gene transfer techniques, on the other hand, inhibits apoptosis in rat smooth muscle cells [29], murine aortic endothelial cells [30]following ischemia–reperfusion. Misao et al. [31]conducted immunohistochemical studies in 37 autopsied human hearts with acute and old myocardial infarction and normal hearts with the use of bcl-2 antibody. They found that bcl-2 protein is expressed in salvaged myocytes during the acute stage of infarction. The expression of bcl-2 may play an important pathophysiological role in the protection from or acceleration of apoptosis of human myocytes after ischemia and/or reperfusion. A recent study [32]showed that P53 not only repressed transcription of bcl-2, but also upregulated the local renin–angiotensin system, including the formation and secretion of Ang II in rat ventricular myocytes. AT1R blocker losartan and Ang II antibody prevented P53-induced apoptosis. Thus, an increase in Ang II or a decrease in bcl-2-protein mediated by P53 triggers apoptosis in rat ventricular myocytes. The present study for the first time shows that anoxia–reoxygenation decreases bcl-2 protein expression in cultured HCAECs, and Ang II further decreases bcl-2 protein expression beyond that caused by anoxia–reoxygenation. Importantly, losartan significantly increased bcl-2 protein expression in HCAECs and blocked the effect of Ang II.

Increase in Fas expression predicts impending apoptosis [11]. Tanaka et al. [11]showed that hypoxia-induced apoptosis is associated with enhanced expression of Fas mRNA in cultured neonatal rat cardiomyocytes. Dong [12]observed concurrent Fas expression and apoptosis by immunohistochemistry in atherosclerotic arterial tissues. Fas positivity was identified mainly in the endothelial cells and to a much smaller extent in the macrophages. The present study for the first time shows that anoxia–reoxygenation increases Fas protein expression in cultured HCAECs. Ang II further enhances Fas protein expression beyond that caused by anoxia–reoxygenation. Losartan markedly decreased Fas protein expression in HCAECs in the presence of Ang II, suggesting that Ang II-mediated increase in Fas protein expression is a result of AT1R activation.

These observations indicate that increase in Fas protein expression and a decrease in cNOS and bcl-2 may play a role in anoxia–reoxygenation and Ang II-induced apoptosis of HCAECs. Importantly, blockade of Ang II-mediated changes by losartan underscores pathogenic role of AT1R activation in apoptosis. These data also confirm the critical role of AT1R activation in the determination of tissue injury caused by anoxia–reoxygenation.

Time for primary review 22 days.

	Acknowledgements

 
This work was supported by a grant-in-aid from the American Heart Association-Florida Affiliate, St. Petersburg, FL, and a Merit Review Award from the VA Center Office.

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