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Angiotensin II-mediated apoptosis on human vascular smooth muscle cells
Journal of Cardiothoracic-Renal Research (2006) 1, 135—139
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/jcrr
Angiotensin II-mediated apoptosis on human vascular smooth muscle cells Hong Song a, Daqing Gao b, Lei Chen b, Koichi Seta a, Joseph S. McLaughlin a, Chiming Wei b,∗ a
Division of Thoracic and Cardiovascular Surgery, Department of Surgery, University of Maryland School of Medicine, Baltimore, MD 21201, United States b Cardiothoracic-Renal Molecular Research Program, Department of Surgery, Johns Hopkins University School of Medicine, 600 N. Wolfe Street/Blalock 1206, Baltimore, MD 21205, United States Received 20 May 2006; accepted 20 May 2006
KEYWORDS Angiotensin II; Apoptosis; Vascular smooth muscle cells; AT-1 receptor; AT-2 receptor
Summary While previous studies demonstrated that angiotensin II is a potent vasoconstrictor and mitogenic factor, the effect of angiotensin II on apoptosis in vascular smooth muscle cells remain controversial. Therefore, the current study was designed to investigate the action of angiotensin II on apoptosis in human vascular smooth muscle cells. Human saphenous vein was obtained from coronary artery bypass surgery (n = 6) and was minced and incubated in the special tissue culture system in the absence or presence of angiotensin II (10−7 M) for 24 h. These studies were repeated with co-incubation of losartan (AT-1 receptor antagonist, 10−6 M) or PD-123319 (AT-2 receptor antagonist, 10−6 M). To detect the in situ DNA fragmentation, TUNEL staining was performed. TUNEL staining demonstrated that angiotensin II increased apoptosis in human vascular smooth muscle cells. This action of angiotensin II was enhanced by losartan and attenuated by PD-123319. Furthermore, co-incubation with both losartan and PD-123319 significantly reduced apoptosis levels. In conclusion, these data demonstrated that angiotensin II has potent apoptotic effect in human vascular smooth muscle cells through both AT-1 and AT-2 receptors. Furthermore, angiotensin II through AT-2 receptor has more potent apoptotic action in human vascular smooth muscle cells. This study indicated that angiotensin II plays an important role in the processes of apoptosis via angiotensin II receptors in human vascular smooth muscle cells. © 2006 Asian-Pacific Cardiothoracic-Renal Association (APCRA). Published by Elsevier Ltd. All rights reserved.
Introduction ∗
Corresponding author. Tel.: +1 410 502 0622; fax: +1 410 502 0078. E-mail address: [email protected] (C. Wei).
Apoptosis of vascular smooth muscle cells has been described in disease such as atherosclerosis, and restenosis after angioplasty and bypass grafting, as well as develop-
1574-0668/$ — see front matter © 2006 Asian-Pacific Cardiothoracic-Renal Association (APCRA). Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.jccr.2006.05.005
136 ment and physiological remodeling of vessels [1—4]. It has been believed that vascular structure is determined in large part by a balance between cell growth and cell death by apoptosis. However, the factors that regulate this balance between vascular smooth muscle cells death and survival remains to be defined. Angiotensin II importantly contributes to the pathobiology of atherosclerosis and vascular disease not only via its role in hypertension but also via its direct effects on vascular smooth muscle cells. Angiotensin II has previously been shown to promote the growth of vascular smooth muscle cells via activation of AT-1 receptor [5—8]. More recent studies have demonstrated that the angiotensin II through AT-1 receptor or AT-2 receptor mediates apoptosis in vascular smooth muscle cells [9,10]. Angiotensin II represents a bifunctional growth factor by simultaneously stimulating proliferative and apoptotic pathways. Based upon these previous studies, we hypothesize that angiotensin II may produce apoptosis in human vascular smooth muscle cells of saphenous vein via angiotensin II receptors. Therefore, the present study was designed to investigate the pathophysiological roles of angiotensin IImediate apoptosis in human vascular smooth muscle cells.
Methods Human subjects and venous tissue incubation Saphenous veins of six patients were obtained intraoperatively during the coronary artery bypass surgery. This study was performed with the approval of the Institutional Review Board of the University of Maryland School of Medicine. The mean age (±S.E.M.) of the patients was 66 ± 2 years (range, 55—77). These patients include five men and one woman. After venous tissue excision, the samples were immediately placed in oxygenated, nominally Ca2+ -free Tyrode solution for transport to the laboratory. The tissues were chopped with scissors into cubic chunks (0.5 mm3 ) in nominally Ca2+ free Tyrode solution (36 ◦ C). The Tyrode solution contained (mmol/L): NaCl 126.0, KCl 5.4, MgCl2 1.0, NaH2 PO4 0.33, glucose 10.0, and HEPES 10.0, pH 7.4. The tissues then were placed in the special tissue culture system with 5% serum and culture medium (Clontech Laboratories, Inc., San Diego, CA) for 24 h. For negative control, we also investigated the TUNEL staining in fresh human saphenous vein tissue without incubation. Venous samples from each patient were divided into seven study groups: (1) control (vehicle group); (2) angiotensin II alone (10−7 M); (3) angiotensin II with losartan (10−6 M, AT-1 receptor antagonist); (4) angiotensin II with PD-123319 (10−6 M, AT-2 receptor antagonist); (5) angiotensin II with both losartan and PD-123319; (6) losartan alone; (7) PD-123319 alone. After incubation, the venous tissues were immediately fixed with 10% buffered formalin for further studies. The venous tissue viability was determined by following methods: (1) staining with anti-alpha-1 anti-trypsin antibody, anti-kappa light chain antibody and anti-lambda light chain antibody; (2) plasma membrane marker ouabainsensitive Na+ , K+ -ATPase activities; (3) RNA quality, judging from 28S/18S ratio of rRNA and GAPDH mRNA; (4) tetrazolium derivative reduction (MTT) assay; (5) electro
H. Song et al. microscopy; (6) TUNEL staining and DNA gel electrophoresis. Our previous studies demonstrated that all of these evaluations were no significant difference between fresh tissue and tissue after 24-h incubation in our special tissue culture system. These evidences established that our tissue culture system is reliable with no significant influence on the results of our experiments.
In situ identification of nuclear DNA fragmentation To detect the DNA fragmentation in situ, nick-end labeling was performed with the ApopTag in situ apoptosis detection kit (Oncor, Gaithersburg, MD). The procedure is based upon the method described previously [1—4,9,10] with minor modifications. Briefly, human venous tissue was fixed in 10% formalin and embedded in paraffin. Serial sections were prepared at a thickness of 5 m. Tissue sections were deparaffinized and treated with 3% hydrogen peroxide. The slides were incubated with 2% H2 O2 for 7 min to inactivate the endogenous peroxidase and covered with 0.3 g/L terminal deoxynucleotidyl transferase (TDT, Boehringer Mannheim) and 0.04 nmol/L biotinylated dUTP (Boehringer Mannheim) in TDT buffer containing 30 mmol/L cobalt chloride for 90 min at 37 ◦ C. The reaction was terminated with buffer containing 30 mmol/L sodium citrate and 300 mmol/L NaCl. The slides were covered with 5% normal goat serum and applied with horseradish peroxidaseconjugated streptavidin (Nichirel). Peroxidase was visualized using the chromogen 3,3 -diaminobenzidine and H2 O2 . Counterstaining was performed with hematoxylin. For negative control to DNA fragmentation labeling, the serial sections were stained without terminal ozynucleotidyl transferase. An average of 1000 nuclei from random fields was analyzed for each data point. The percentage of apoptotic cells was determined by means of an apoptotic index. The apoptotic index (percentage of apoptotic nuclei) was calculated as (apoptotic nuclei/total nuclei) × 100%. Apoptotic index of 0.5 or less was considered to indicate the absence of apoptosis. Sample identities were concealed during scoring, and at least three samples were scored per group. The nuclei without DNA fragmentation stained blue as a result of counterstaining with hematoxylin. The nuclei with DNA fragmentation stained brown color, and nuclei without DNA fragmentation had clear blue nuclear regions.
Statistics Results of the quantitative studies are expressed as means ± S.E.M. Statistical significance (p < 0.05) in comparisons between two measurements and among groups was determined by the two-tailed Student’s t-test and by analysis of variance with the Bonferroni method, respectively.
Results TUNEL staining demonstrated that angiotensin II increased apoptosis in human vascular smooth muscle cells. The apoptotic index in angiotensin II group (Fig. 1, TUNEL staining score: 27.6 ± 4.1%) was significantly increased compared
Angiotensin II-mediated apoptosis on human vascular smooth muscle cells
137
Figure 1 In contrast, TUNEL study in angiotensin II group (10−7 M) illustrates significantly increase positive TUNEL staining in nuclear (arrow) of vascular smooth muscle cells (×1000).
Figure 3 In angiotensin II (10−7 M) plus losartan (10−6 M, angiotensin II type 1 receptor [AT-1] antagonist) group, the apoptotic level (TUNEL staining) was further increased (arrow) compared with that in angiotensin II group in vascular smooth muscle cells (×1000).
with that in control group (Fig. 2, TUNEL staining score: 0.6 ± 0.1%, p < 0.05 versus angiotensin II group). The apoptotic effect of angiotensin II was enhanced in angiotensin II plus losartan group (Fig. 3, TUNEL staining score: 34.2 ± 6.2%, p < 0.05 versus angiotensin II group) and attenuated in angiotensin II plus PD-123319 group (Fig. 4, TUNEL staining score: 17.6 ± 3.2%, p < 0.05 versus angiotensin
II plus losartan group). Furthermore, co-incubation with both AT-1 and AT-2 receptor blockade (angiotensin II plus losartan and PD-123319 group) significantly decreased the apoptotic labeling index in vascular smooth muscle cells (Fig. 5, TUNEL staining score: 5.5 ± 0.8%, p < 0.05 versus
Figure 2 In situ terminal deoxymucleotidyl transferase dUTP nick end labeling (TUNEL study) demonstrates very mild positive TUNEL staining in human vascular smooth muscle cells in control group (×1000).
Figure 4 In contrast, in situ terminal deoxymucleotidyl transferase dUTP nick end labeling (TUNEL study) in angiotensin II plus PD-123319 (angiotensin II type 2 receptor [AT-2] antagonist) group (10−6 M) illustrates markedly decrease positive TUNEL staining in nuclear (arrow) of vascular smooth muscle cells (×1000).
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Discussion
Figure 5 In angiotensin II (10−7 M) plus losartan (10−6 M) and PD-123319 (10−6 M) group, the apoptotic level (TUNEL staining) was significantly decreased compared with angiotensin II group in vascular smooth muscle cells (×1000).
angiotensin II group, angiotensin II plus losartan group, and angiotensin II plus PD-123319 group). Fig. 6 summarized the actions of angiotensin II on vascular smooth muscle cell apoptosis and the effects of losartan and PD-123319 on angiotensin II-mediated apoptosis in vascular smooth muscle cells.
Figure 6 The average of TUNEL staining index (%) in each study group is shown in this figure. Angiotensin II significantly increased apoptosis in human vascular smooth muscle cells. Coincubation of angiotensin II with losartan (L) further enhanced the apoptotic effect of angiotensin II in human vascular smooth muscle cells. In contract, co-incubation of angiotensin II with PD-123319 (PD) attenuated angiotensin II-mediated apoptotic effects. Furthermore, co-incubation of angiotensin II with both losartan and PD-123319 significantly decreased the apoptotic index in vascular smooth muscle cells.
The present study demonstrated that angiotensin II has potent apoptotic effect in human vascular smooth muscle cells through both AT-1 and AT-2 receptors. Furthermore, angiotensin II through AT-2 receptor has more potent apoptotic action in human vascular smooth muscle cells. This study indicated that angiotensin II plays an important role in the processes of apoptosis via angiotensin II receptors in human vascular smooth muscle cells. Apoptosis is involved in many cardiovascular diseases. There are a number of genes in the regulation of apoptosis. These genes include apoptosis activators such as p53 [11], c-myc [12], bax [13], p21-WAF [14], and genes that inhibit apoptosis, such as bcl-2 gene family [15]. The mechanisms of apoptosis include p53-dependent pathway and p53independent pathway. These genes have been reported to be responsible for the modulation of certain stress-induced apoptosis and cell cycle arrest. Blockade of the AT-1 receptor by the selective antagonist losartan might result in the shunting of angiotensin II to the AT-2 receptor. Although the functional effects mediated by the AT-2 receptor remain unclear, there is increasing evidence that in some tissues the AT-2 receptor may activate pathways that result in inhibition of cell growth [16] or even stimulation of apoptotic pathways [17,18]. More recent studies have demonstrated that the AT-2 receptor mediates apoptosis in PC12W cells [17], vascular smooth muscle cells [9] and cardiomyocytes [19]. Taken together, the enhancement of smooth muscle cell apoptosis in response to the AT-1 receptor antagonist losartan in our investigation might be due to the apoptotic action of AT-2 receptor activation. While the mechanisms of proapoptotic effects acting via the AT-2 receptor in vascular smooth muscle cell have not been fully understood, there is evidence that apoptosis may be induced by dephosphorylation of mitogen-activated protein kinase secondary to AT-2 receptor stimulation in PC12W (rat pheochromocytoma) cells and R3T3 (mouse fibroblast) cells [17]. Recent in vitro studies demonstrated that selective stimulation of AT-2 receptor facilitated serum-deprivationinduced apoptosis in vascular smooth muscle cells [9]. Moreover, AT-1 receptor stimulation activated extracellular signal-regulated kinase (ERK), whereas the AT-2 receptor stimulation inhibited the activation of ERK [9]. These studies suggest that AT-1 and AT-2 receptors exert counteracting effects on extracellular signal-regulated kinase activation and consequently vascular smooth muscle cell apoptosis and differential expression of these receptor may participate in vascular development and vascular remodeling. On the other hand, recent in vivo study demonstrated that chronic administration of losartan or PD-123319 with angiotensin II result in increasing of apoptosis and apoptosisrelated gene Bax and caspase-3 expression in rat aortic smooth muscle cells [10]. This study reported that both AT-1 or AT-2 receptor stimulation in vivo caused enhancement of apoptosis in the media of aortic blood vessels. The increase in systolic blood pressure and aortic growth induced by angiotensin II infusion was completely inhibited by losartan, whereas co-infusion of PD-123319 resulted in a greater increases in systolic blood pressure and aortic smooth muscle cell proliferation compared with angiotensin
Angiotensin II-mediated apoptosis on human vascular smooth muscle cells II alone group [10]. Therefore, the AT-1 receptor-mediated vascular smooth muscle cell apoptosis may occur secondary to vascular smooth muscle cell proliferation and remodeling. Furthermore, both bax and caspase-3 participate in the pathways of apoptosis triggered by in vivo AT-1 receptor stimulation. In present study, we demonstrated that both AT1 and AT-2 receptor stimulation result in increasing apoptosis in human vascular smooth muscle cells. The apoptotic action of AT-2 receptor is more potent compared with AT-1 receptor. Therefore, the AT-1 and AT-2 receptors may have distinct effect on receptors-mediated intracellular downstream signaling pathways and in the regulation of proliferation and apoptosis in vascular smooth muscle cells. In summary, the current study reported that potent apoptotic effect of angiotensin II in human vascular smooth muscle cells mediated by both AT-1 and AT-2 receptor stimulation. Furthermore, AT-2 receptor activation result in the enhancement of apoptosis in vascular smooth muscle cells compared with AT-1 receptor stimulation. These data suggest that angiotensin II via its receptors play an important role in regulation of apoptosis in vascular smooth muscle cells.
[6]
[7]
[8]
[9]
[10]
[11]
Acknowledgments This work was supported in part by grants from National Heart, Lung and Blood Institute (HL03174 and HL61299, C. Wei), Merck & Co., Inc., American Heart Association MidAtlantic Affiliate, National Kidney Foundation and the University of Maryland School of Medicine.
[12]
[13] [14] [15]
References [16] [1] Han D, Haudenschild C, Hong M, Tinkle B, Leon M, Liau G. Evidence for apoptosis in human atherosclerosis and in a rat vascular injury model. Am J Path 1995;147:267—77. [2] Schwartz S, Bennett M. Death by any other name. Am J Path 1995;147:229—34. [3] Bennett MR, Evan GI, Schwartz SM. Apoptosis of human vascular smooth muscle cells derived from normal vessels and coronary atherosclerotic plaques. J Clin Invest 1995;95:2266—74. [4] Bennett M, Littlewood T, Schwartz S, Weissberg P. Increased sensitivity of human vascular smooth muscle cells from atherosclerotic plaque to p53-mediated apoptosis. Circ Res 1997;81:591—9. [5] Deguchi J, Makuuchi M, Nakaoka T, Collins T, Takuwa Y. Angiotensin II stimulates platelet-derived growth factor-B
[17]
[18]
[19]
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chain expression in newborn rat vascular smooth muscle cells and neointimal cells through Ras, extracellular signalregulated protein kinase, and c-Jun N-terminal protein kinase mechanisms. Circ Res 1999;85:565—74. Ford CM, Li S, Pickering JG. Angiotensin II stimulates collagen synthesis in human vascular smooth muscle cells. Involvement of the AT(1) receptor, transforming growth factor-beta, and tyrosine phosphorylation. Arterioscler Thromb Vasc Biol 1999;19(8):1843—51. Sung CP, Arleth AJ, Storer BL, Ohlstein EH. Angiotensin type 1 receptors mediate smooth muscle proliferation and endothelin biosynthesis in rat vascular smooth muscle. J Pharmacol Exp Ther 1994;271:429—37. Geisterfer AA, Peach MJ, Owens GK. Angiotensin II induces hypertrophy, not hyperplasia, of cultured rat aortic smooth muscle cells. Circ Res 1988;62:749—56. Yamada T, Akishita M, Pollman MJ, Gibbons GH, Dzau VJ, Horiuchi M. Angiotensin II type 2 receptor mediates vascular smooth muscle cell apoptosis and antagonizes angiotensin II type 1 receptor action: an in vitro gene transfer study. Life Sci 1998;63:289—95. Diep QN, Li JS, Schiffrin EL. In vivo study AT(1) and AT(2) angiotensin receptors in apoptosis in rat blood vessels. Hypertension 1999;34:617—24. Levine AJ, Momand J, Finlay CA. The p53 tumor suppressor gene. Nature 1991;351:453—6. Fanidi A, Harrington EA, Evan GI. Cooperative interaction between c-myc and bac-2 proto-oncogenes. Nature 1992;359:554—6. Elledge SJ. Cell cycle checkpoints: preventing an identity crisis. Science 1996;274:1664—72. El-Deiry WS, Tokino T, Velculescu VE, et al. WAF1, a potential mediator of p53 tumor suppression. Cell 1993;75:817—25. Garcia I, Martinou I, Tsujimoto Y, Martinou JC. Prevention of programmed cell death of sympathetic neurons by the bcl-2 proto-oncogene. Science 1992;258:302—4. Stoll M, Steckelings U, Paul M, Bottari S, Metzger R, Unger T. The angiotensin AT2-receptor mediates inhibition of cell proliferation in coronary endothelial cells. J Clin Invest 1995;95:651—7. Yamada T, Toriuchi M, Dzau VJ. Angiotensin II type 2 receptor mediates programmed cell death. Proc Natl Acad Sci USA 1996;93:156—60. Tanaka M, Ohnishi J, Ozawa Y, Sugimoto M, Usuki S, Naruse M, et al. Characterization of angiotensin II receptor type 2 during differentiation and apoptosis of rat ovarian cultured granulosa cells. Biochem Biophys Res Commun 1995;207:593—8. Daviet L, Horiuchi M, Hayashida W, Lehtonen JYA, Bishopric NH, Dzau VJ. Antagonistic actions of type-1 and -2 angiotensin II receptors on rat cardiomyocyte apoptosis. Circulation 1998;98:1—741.
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/jcrr
Angiotensin II-mediated apoptosis on human vascular smooth muscle cells Hong Song a, Daqing Gao b, Lei Chen b, Koichi Seta a, Joseph S. McLaughlin a, Chiming Wei b,∗ a
Division of Thoracic and Cardiovascular Surgery, Department of Surgery, University of Maryland School of Medicine, Baltimore, MD 21201, United States b Cardiothoracic-Renal Molecular Research Program, Department of Surgery, Johns Hopkins University School of Medicine, 600 N. Wolfe Street/Blalock 1206, Baltimore, MD 21205, United States Received 20 May 2006; accepted 20 May 2006
KEYWORDS Angiotensin II; Apoptosis; Vascular smooth muscle cells; AT-1 receptor; AT-2 receptor
Summary While previous studies demonstrated that angiotensin II is a potent vasoconstrictor and mitogenic factor, the effect of angiotensin II on apoptosis in vascular smooth muscle cells remain controversial. Therefore, the current study was designed to investigate the action of angiotensin II on apoptosis in human vascular smooth muscle cells. Human saphenous vein was obtained from coronary artery bypass surgery (n = 6) and was minced and incubated in the special tissue culture system in the absence or presence of angiotensin II (10−7 M) for 24 h. These studies were repeated with co-incubation of losartan (AT-1 receptor antagonist, 10−6 M) or PD-123319 (AT-2 receptor antagonist, 10−6 M). To detect the in situ DNA fragmentation, TUNEL staining was performed. TUNEL staining demonstrated that angiotensin II increased apoptosis in human vascular smooth muscle cells. This action of angiotensin II was enhanced by losartan and attenuated by PD-123319. Furthermore, co-incubation with both losartan and PD-123319 significantly reduced apoptosis levels. In conclusion, these data demonstrated that angiotensin II has potent apoptotic effect in human vascular smooth muscle cells through both AT-1 and AT-2 receptors. Furthermore, angiotensin II through AT-2 receptor has more potent apoptotic action in human vascular smooth muscle cells. This study indicated that angiotensin II plays an important role in the processes of apoptosis via angiotensin II receptors in human vascular smooth muscle cells. © 2006 Asian-Pacific Cardiothoracic-Renal Association (APCRA). Published by Elsevier Ltd. All rights reserved.
Introduction ∗
Corresponding author. Tel.: +1 410 502 0622; fax: +1 410 502 0078. E-mail address: [email protected] (C. Wei).
Apoptosis of vascular smooth muscle cells has been described in disease such as atherosclerosis, and restenosis after angioplasty and bypass grafting, as well as develop-
1574-0668/$ — see front matter © 2006 Asian-Pacific Cardiothoracic-Renal Association (APCRA). Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.jccr.2006.05.005
136 ment and physiological remodeling of vessels [1—4]. It has been believed that vascular structure is determined in large part by a balance between cell growth and cell death by apoptosis. However, the factors that regulate this balance between vascular smooth muscle cells death and survival remains to be defined. Angiotensin II importantly contributes to the pathobiology of atherosclerosis and vascular disease not only via its role in hypertension but also via its direct effects on vascular smooth muscle cells. Angiotensin II has previously been shown to promote the growth of vascular smooth muscle cells via activation of AT-1 receptor [5—8]. More recent studies have demonstrated that the angiotensin II through AT-1 receptor or AT-2 receptor mediates apoptosis in vascular smooth muscle cells [9,10]. Angiotensin II represents a bifunctional growth factor by simultaneously stimulating proliferative and apoptotic pathways. Based upon these previous studies, we hypothesize that angiotensin II may produce apoptosis in human vascular smooth muscle cells of saphenous vein via angiotensin II receptors. Therefore, the present study was designed to investigate the pathophysiological roles of angiotensin IImediate apoptosis in human vascular smooth muscle cells.
Methods Human subjects and venous tissue incubation Saphenous veins of six patients were obtained intraoperatively during the coronary artery bypass surgery. This study was performed with the approval of the Institutional Review Board of the University of Maryland School of Medicine. The mean age (±S.E.M.) of the patients was 66 ± 2 years (range, 55—77). These patients include five men and one woman. After venous tissue excision, the samples were immediately placed in oxygenated, nominally Ca2+ -free Tyrode solution for transport to the laboratory. The tissues were chopped with scissors into cubic chunks (0.5 mm3 ) in nominally Ca2+ free Tyrode solution (36 ◦ C). The Tyrode solution contained (mmol/L): NaCl 126.0, KCl 5.4, MgCl2 1.0, NaH2 PO4 0.33, glucose 10.0, and HEPES 10.0, pH 7.4. The tissues then were placed in the special tissue culture system with 5% serum and culture medium (Clontech Laboratories, Inc., San Diego, CA) for 24 h. For negative control, we also investigated the TUNEL staining in fresh human saphenous vein tissue without incubation. Venous samples from each patient were divided into seven study groups: (1) control (vehicle group); (2) angiotensin II alone (10−7 M); (3) angiotensin II with losartan (10−6 M, AT-1 receptor antagonist); (4) angiotensin II with PD-123319 (10−6 M, AT-2 receptor antagonist); (5) angiotensin II with both losartan and PD-123319; (6) losartan alone; (7) PD-123319 alone. After incubation, the venous tissues were immediately fixed with 10% buffered formalin for further studies. The venous tissue viability was determined by following methods: (1) staining with anti-alpha-1 anti-trypsin antibody, anti-kappa light chain antibody and anti-lambda light chain antibody; (2) plasma membrane marker ouabainsensitive Na+ , K+ -ATPase activities; (3) RNA quality, judging from 28S/18S ratio of rRNA and GAPDH mRNA; (4) tetrazolium derivative reduction (MTT) assay; (5) electro
H. Song et al. microscopy; (6) TUNEL staining and DNA gel electrophoresis. Our previous studies demonstrated that all of these evaluations were no significant difference between fresh tissue and tissue after 24-h incubation in our special tissue culture system. These evidences established that our tissue culture system is reliable with no significant influence on the results of our experiments.
In situ identification of nuclear DNA fragmentation To detect the DNA fragmentation in situ, nick-end labeling was performed with the ApopTag in situ apoptosis detection kit (Oncor, Gaithersburg, MD). The procedure is based upon the method described previously [1—4,9,10] with minor modifications. Briefly, human venous tissue was fixed in 10% formalin and embedded in paraffin. Serial sections were prepared at a thickness of 5 m. Tissue sections were deparaffinized and treated with 3% hydrogen peroxide. The slides were incubated with 2% H2 O2 for 7 min to inactivate the endogenous peroxidase and covered with 0.3 g/L terminal deoxynucleotidyl transferase (TDT, Boehringer Mannheim) and 0.04 nmol/L biotinylated dUTP (Boehringer Mannheim) in TDT buffer containing 30 mmol/L cobalt chloride for 90 min at 37 ◦ C. The reaction was terminated with buffer containing 30 mmol/L sodium citrate and 300 mmol/L NaCl. The slides were covered with 5% normal goat serum and applied with horseradish peroxidaseconjugated streptavidin (Nichirel). Peroxidase was visualized using the chromogen 3,3 -diaminobenzidine and H2 O2 . Counterstaining was performed with hematoxylin. For negative control to DNA fragmentation labeling, the serial sections were stained without terminal ozynucleotidyl transferase. An average of 1000 nuclei from random fields was analyzed for each data point. The percentage of apoptotic cells was determined by means of an apoptotic index. The apoptotic index (percentage of apoptotic nuclei) was calculated as (apoptotic nuclei/total nuclei) × 100%. Apoptotic index of 0.5 or less was considered to indicate the absence of apoptosis. Sample identities were concealed during scoring, and at least three samples were scored per group. The nuclei without DNA fragmentation stained blue as a result of counterstaining with hematoxylin. The nuclei with DNA fragmentation stained brown color, and nuclei without DNA fragmentation had clear blue nuclear regions.
Statistics Results of the quantitative studies are expressed as means ± S.E.M. Statistical significance (p < 0.05) in comparisons between two measurements and among groups was determined by the two-tailed Student’s t-test and by analysis of variance with the Bonferroni method, respectively.
Results TUNEL staining demonstrated that angiotensin II increased apoptosis in human vascular smooth muscle cells. The apoptotic index in angiotensin II group (Fig. 1, TUNEL staining score: 27.6 ± 4.1%) was significantly increased compared
Angiotensin II-mediated apoptosis on human vascular smooth muscle cells
137
Figure 1 In contrast, TUNEL study in angiotensin II group (10−7 M) illustrates significantly increase positive TUNEL staining in nuclear (arrow) of vascular smooth muscle cells (×1000).
Figure 3 In angiotensin II (10−7 M) plus losartan (10−6 M, angiotensin II type 1 receptor [AT-1] antagonist) group, the apoptotic level (TUNEL staining) was further increased (arrow) compared with that in angiotensin II group in vascular smooth muscle cells (×1000).
with that in control group (Fig. 2, TUNEL staining score: 0.6 ± 0.1%, p < 0.05 versus angiotensin II group). The apoptotic effect of angiotensin II was enhanced in angiotensin II plus losartan group (Fig. 3, TUNEL staining score: 34.2 ± 6.2%, p < 0.05 versus angiotensin II group) and attenuated in angiotensin II plus PD-123319 group (Fig. 4, TUNEL staining score: 17.6 ± 3.2%, p < 0.05 versus angiotensin
II plus losartan group). Furthermore, co-incubation with both AT-1 and AT-2 receptor blockade (angiotensin II plus losartan and PD-123319 group) significantly decreased the apoptotic labeling index in vascular smooth muscle cells (Fig. 5, TUNEL staining score: 5.5 ± 0.8%, p < 0.05 versus
Figure 2 In situ terminal deoxymucleotidyl transferase dUTP nick end labeling (TUNEL study) demonstrates very mild positive TUNEL staining in human vascular smooth muscle cells in control group (×1000).
Figure 4 In contrast, in situ terminal deoxymucleotidyl transferase dUTP nick end labeling (TUNEL study) in angiotensin II plus PD-123319 (angiotensin II type 2 receptor [AT-2] antagonist) group (10−6 M) illustrates markedly decrease positive TUNEL staining in nuclear (arrow) of vascular smooth muscle cells (×1000).
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Discussion
Figure 5 In angiotensin II (10−7 M) plus losartan (10−6 M) and PD-123319 (10−6 M) group, the apoptotic level (TUNEL staining) was significantly decreased compared with angiotensin II group in vascular smooth muscle cells (×1000).
angiotensin II group, angiotensin II plus losartan group, and angiotensin II plus PD-123319 group). Fig. 6 summarized the actions of angiotensin II on vascular smooth muscle cell apoptosis and the effects of losartan and PD-123319 on angiotensin II-mediated apoptosis in vascular smooth muscle cells.
Figure 6 The average of TUNEL staining index (%) in each study group is shown in this figure. Angiotensin II significantly increased apoptosis in human vascular smooth muscle cells. Coincubation of angiotensin II with losartan (L) further enhanced the apoptotic effect of angiotensin II in human vascular smooth muscle cells. In contract, co-incubation of angiotensin II with PD-123319 (PD) attenuated angiotensin II-mediated apoptotic effects. Furthermore, co-incubation of angiotensin II with both losartan and PD-123319 significantly decreased the apoptotic index in vascular smooth muscle cells.
The present study demonstrated that angiotensin II has potent apoptotic effect in human vascular smooth muscle cells through both AT-1 and AT-2 receptors. Furthermore, angiotensin II through AT-2 receptor has more potent apoptotic action in human vascular smooth muscle cells. This study indicated that angiotensin II plays an important role in the processes of apoptosis via angiotensin II receptors in human vascular smooth muscle cells. Apoptosis is involved in many cardiovascular diseases. There are a number of genes in the regulation of apoptosis. These genes include apoptosis activators such as p53 [11], c-myc [12], bax [13], p21-WAF [14], and genes that inhibit apoptosis, such as bcl-2 gene family [15]. The mechanisms of apoptosis include p53-dependent pathway and p53independent pathway. These genes have been reported to be responsible for the modulation of certain stress-induced apoptosis and cell cycle arrest. Blockade of the AT-1 receptor by the selective antagonist losartan might result in the shunting of angiotensin II to the AT-2 receptor. Although the functional effects mediated by the AT-2 receptor remain unclear, there is increasing evidence that in some tissues the AT-2 receptor may activate pathways that result in inhibition of cell growth [16] or even stimulation of apoptotic pathways [17,18]. More recent studies have demonstrated that the AT-2 receptor mediates apoptosis in PC12W cells [17], vascular smooth muscle cells [9] and cardiomyocytes [19]. Taken together, the enhancement of smooth muscle cell apoptosis in response to the AT-1 receptor antagonist losartan in our investigation might be due to the apoptotic action of AT-2 receptor activation. While the mechanisms of proapoptotic effects acting via the AT-2 receptor in vascular smooth muscle cell have not been fully understood, there is evidence that apoptosis may be induced by dephosphorylation of mitogen-activated protein kinase secondary to AT-2 receptor stimulation in PC12W (rat pheochromocytoma) cells and R3T3 (mouse fibroblast) cells [17]. Recent in vitro studies demonstrated that selective stimulation of AT-2 receptor facilitated serum-deprivationinduced apoptosis in vascular smooth muscle cells [9]. Moreover, AT-1 receptor stimulation activated extracellular signal-regulated kinase (ERK), whereas the AT-2 receptor stimulation inhibited the activation of ERK [9]. These studies suggest that AT-1 and AT-2 receptors exert counteracting effects on extracellular signal-regulated kinase activation and consequently vascular smooth muscle cell apoptosis and differential expression of these receptor may participate in vascular development and vascular remodeling. On the other hand, recent in vivo study demonstrated that chronic administration of losartan or PD-123319 with angiotensin II result in increasing of apoptosis and apoptosisrelated gene Bax and caspase-3 expression in rat aortic smooth muscle cells [10]. This study reported that both AT-1 or AT-2 receptor stimulation in vivo caused enhancement of apoptosis in the media of aortic blood vessels. The increase in systolic blood pressure and aortic growth induced by angiotensin II infusion was completely inhibited by losartan, whereas co-infusion of PD-123319 resulted in a greater increases in systolic blood pressure and aortic smooth muscle cell proliferation compared with angiotensin
Angiotensin II-mediated apoptosis on human vascular smooth muscle cells II alone group [10]. Therefore, the AT-1 receptor-mediated vascular smooth muscle cell apoptosis may occur secondary to vascular smooth muscle cell proliferation and remodeling. Furthermore, both bax and caspase-3 participate in the pathways of apoptosis triggered by in vivo AT-1 receptor stimulation. In present study, we demonstrated that both AT1 and AT-2 receptor stimulation result in increasing apoptosis in human vascular smooth muscle cells. The apoptotic action of AT-2 receptor is more potent compared with AT-1 receptor. Therefore, the AT-1 and AT-2 receptors may have distinct effect on receptors-mediated intracellular downstream signaling pathways and in the regulation of proliferation and apoptosis in vascular smooth muscle cells. In summary, the current study reported that potent apoptotic effect of angiotensin II in human vascular smooth muscle cells mediated by both AT-1 and AT-2 receptor stimulation. Furthermore, AT-2 receptor activation result in the enhancement of apoptosis in vascular smooth muscle cells compared with AT-1 receptor stimulation. These data suggest that angiotensin II via its receptors play an important role in regulation of apoptosis in vascular smooth muscle cells.
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[10]
[11]
Acknowledgments This work was supported in part by grants from National Heart, Lung and Blood Institute (HL03174 and HL61299, C. Wei), Merck & Co., Inc., American Heart Association MidAtlantic Affiliate, National Kidney Foundation and the University of Maryland School of Medicine.
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