Matrix metalloproteinase 2 is suppressed by trapidil, a CD40 CD40 ligand pathway inhibitor, in human abdominal aortic aneurysm wall

Transcription

1 Matrix metalloproteinase 2 is suppressed by trapidil, a CD40 CD40 ligand pathway inhibitor, in human abdominal aortic aneurysm wall Hirotaka Nagashima, MD, PhD, a Yoshikazu Aoka, MD, a Yasunari Sakomura, MD, PhD, a Kenta Uto, MD, a Akiko Sakuta, MD, a Shigeyuki Aomi, MD, PhD, b Hiromi Kurosawa, MD, PhD, b Nobuhisa Hagiwara, MD, PhD, b Masatoshi Kawana, MD, PhD, a and Hiroshi Kasanuki, MD, PhD, a Tokyo, Japan Background: The activation of inflammatory cells and the production of matrix metalloproteinases (MMPs) are important in the pathogenesis of abdominal aortic aneurysm (AAA). Previous studies have demonstrated that the antiplatelet agent trapidil has multiple actions, including suppression of MMP expression through the inhibition of the CD40 CD40 ligand (CD40-CD40L) pathway in cultured cells. A recent clinical study suggested that trapidil might have functions beyond its antiplatelet action. Methods and results: In the present study, we performed immunohistochemical analysis and semiquantitative reverse transcription polymerase chain reaction to evaluate the effect of trapidil on the production of MMPs in cultured aortic tissues from patients with infrarenal AAA (n 9) and control patients with aortoiliac occlusive disease (n 7). The tissue concentrations of both MMP-2 and MMP-9 were significantly higher in AAA walls than in control aortic walls. Both trapidil and an anti-cd154 (CD40L) antibody significantly suppressed the protein production and mrna expression of MMP-2 but did not inhibit those of MMP-9 in organ cultures of AAA wall specimens. MMP-9 was produced by macrophages and a lot of neutrophils in AAA tissues, whereas MMP-2 was derived from macrophages. CD40 was expressed on macrophages but not on neutrophils, and this expression could explain the differential effect of trapidil on the production of MMP-2 and MMP-9. Conclusions: Trapidil, a CD40-CD40L pathway inhibitor, suppressed mrna expression and protein production of MMP-2 in AAA tissues, suggesting a potential therapeutic approach for the prevention or treatment of AAA. (J Vasc Surg 2004;39: ) Abdominal aortic aneurysm (AAA) is a potentially fatal disorder that affects 2% to 9% of the population older than 65 years. 1,2 Although elective surgical repair or endovascular stent grafting is effective in preventing death from ruptured AAA, there is a conspicuous absence of alternative noninvasive therapeutic strategies for this disease. 3 Because human aortic aneurysms are characterized by destructive remodeling of the aortic wall, investigations have emphasized the mechanism underlying the degradation of matrix proteins in aneurysm wall. 4,5 Several reports have demonstrated that matrix metalloproteinases (MMPs) contribute to the formation and progression of AAA. 6-8 Particularly, MMP-2 and MMP-9 have been demonstrated to be up-regulated in vascular smooth muscle cells from patients with AAA, 9,10 and MMP-9 has been reported to be related to the size or natural history of AAA. 11,12 A recent study using MMP-2 deficient mice and MMP-9 deficient mice showed that two MMPs, MMP-2 and MMP-9, work in concert to produce AAA. 13 These observations have led From the Departments of Cardiology a and Cardiovascular Surgery, b The Heart Institute of Japan, Tokyo Women s Medical University. Competition of interest: none. Reprint requests: Hirotaka Nagashima, MD, Department of Cardiology, The Heart Institute of Japan, Tokyo Women s Medical University, 8-l Kawada-cho, Shinjuku-ku, Tokyo , Japan ( mnagasih@hij.twmu.ac.jp). Published online Nov 10, /$30.00 Copyright 2003 by The Society for Vascular Surgery. doi: /j.jvs to the speculation that MMP-2 and/or MMP-9 might be essential for the development of AAA and, thus, might be a possible key target for pharmacotherapy. 14,15 An antiplatelet drug, trapidil, has been shown to have numerous effects, and it suppresses the in vitro expression of various inflammatory molecules, including MMPs by way of the CD40 CD40 ligand (CD40-CD40L) pathway. 16,17 The CD40-CD40L pathway has been reported to play a central role in the inflammatory aspect of atherosclerotic plaque formation, progression, and subsequent rupture by suppressing MMP activity. 18,19 In fact, a multicenter clinical trial in Japan has shown that trapidil might have more beneficial effect than aspirin on cardiovascular events after myocardial infarction. 20 Therefore, we hypothesized that trapidil might have an inhibitory effect on the production of MMP-2 and/or MMP-9 in AAA tissues and might be a potential therapeutic tool for this disease. 14,15 To address this hypothesis, we investigated the concentrations of MMP-2 and MMP-9 in aortic tissues from patients with AAA and control patients, assessed the effect of trapidil on the production of these MMPs by organ cultures of AAA wall tissues, and performed an immunohistochemical analysis of MMP-2 and MMP-9. METHODS Subjects. Nine male patients (aged years) with infrarenal AAA and 7 control male patients (aged years) with aortoiliac occlusive disease were enrolled 447

2 448 Nagashima et al February 2004 Fig 1. Trapidil significantly reduced the levels of active MMP-2 in a concentration-dependent manner in organ cultures (A) but did not alter that of MMP-9 (B). Value of the trend test, P.001. Fig 2. Effect of trapidil (10 mol/l), anti-cd154 (100 mol/ L), or aspirin (10 mol/l) on active MMP-2 production in AAA tissues. Both trapidil and anti-cd154 antibody inhibited MMP-2 production. *P.01 vs lane 1. in the present study at Tokyo Women s Medical University Hospital. Mean value of the maximum AAA diameter was 5.8 cm. None of the patients were given antiplatelet drugs for at least 1 week before surgery. AAA specimens were obtained from the central portion of the aneurysmal aorta of patients with AAA, and control specimens were collected from the proximal portion of the diseased aorta of patients with aortoiliac occlusive disease during elective surgery. After the specimens were harvested, portions of each were immediately placed into phosphate-buffered saline (PBS; ph 7.2) for organ culture, 4% paraformaldehyde for immunohistochemical analysis, or stored in liquid nitrogen for the preparation of protein and mrna. This study was approved by the human ethics committee of our institute, and all subject patients gave written informed consent. Organ culture of aortic wall specimens. Each specimen was cut into several small pieces (approximately 0.5 cm 3 ) and was incubated with culture medium for vascular smooth muscle cells (VSMCs; HuMedia-SG2; Kurabou, Osaka, Japan) containing various concentrations of trapidil (1-100 mol/l) or vehicle in an atmosphere of 95% O 2 /5% CO 2 for 48 hours at 37 C, as described previously. 15,21 Anti-CD154 (CD40L) blocking monoclonal antibody (10 mol/l, Sigma, St Louis, Mo) or aspirin (100 mol/l, Sigma) was also added to some of the cultures. After 48 hours of culture, total RNA and total protein were prepared from each sample. Measurement of the tissue concentrations of MMP-2, MMP-9, TIMP-1, and TIMP-2. The aortic tissue levels of total MMP-2, MMP-9, tissue inhibitor of metalloproteinase 1 (TIMP-1), and TIMP-2 were examined by enzyme immunoassay (EIA; Fuji Chemical Ltd, Toyama, Japan) with the use of antibodies directed against human MMP-2, human MMP-9, human TIMP-1, and human TIMP-2. Active MMP-2 and active MMP-9 were detected through detection enzyme and the subsequent cleavage of its substrate with the use of the Biotrak MMP-2 activity assay system and the Biotrak MMP-9 activity assay system (both from Amersham, Arlington Heights, Ill). Reverse transcription polymerase chain reaction. Total RNA was isolated from aortic specimens using TRIzol reagent (Gibco BRL, Grand Island, NY), and reverse transcription (RT) of an aliquot (5 g) of the total RNA sample was done by using reverse transcriptase (Superscript II; Gibco BRL). Then polymerase chain reaction (PCR) of the complementary DNA to detect the human MMP-2 and MMP-9 genes was performed by a programmed cycle of 94 C for 30 seconds (denaturation),

3 Volume 39, Number 2 Nagashima et al 449 Fig 3. Representative pattern of semiquantitative RT-PCR for MMP-2 and MMP-9. Trapidil suppressed MMP-2 mrna expression but not MMP-9 mrna expression. 58 C for 30 seconds (annealing), and 72 C for 45 seconds (polymerization). As an internal control, co-amplification of glyceraldehyde phosphate dehydrogenase (GAPDH) was performed. The sequences of the oligonucleotide primers used and the expected size are shown in the Table. The number of PCR cycles was 35, and products were analyzed on agarose gels. To determine whether the PCR was in the linear range, various amounts of total RNA were used to amplify each gene. Semiquantification of mrna expression was achieved by densitometric analysis by using National Institutes of Health (NIH) image analysis software. This analysis confirmed the linear amplification of RNA at amounts from 0.1 to 5.0 g (data not shown), indicating that RT-PCR was performed within the linear range in the present study. Immunohistochemical assays. We used hematoxylin and eosin staining to assess the overall tissue architecture. Immunohistochemistry was performed with a labeled streptavidin biotin (LSAB) kit (Dako, Glostrup, Denmark), and mouse monoclonal antibodies were directed against human T cells (human CD68; Dako), human macrophages (human CD45RO; Dako), human neutrophils (human neutrophil elastase; BD Pharmingen, San Diego, Calif), human MMP-2 (Fuji Chemical Ltd), human MMP-9 (Fuji Chemical Ltd), and human CD 40 (Santa Cruz, Santa Cruz, Calif). The investigators who performed the histologic evaluation were blinded to the clinical data. Statistical analysis. Analyses were performed with SAS System 8e software (SAS Institute Inc, Cary, NC). Results are presented as the mean SD. The normality of the distribution of data was evaluated by the Shapiro-Wilks one-sample test, and the F test was used to assess the homogeneity of variance. The Student t test was used for the comparison of continuous data. A simple linear regression model was used to perform the trend test. One-way analysis of variance was used to test for significant differences between the groups, and Dunnett multiple comparison method was applied when appropriate. A two-tailed P.05 was considered to indicate statistical significance. RESULTS Production of MMP-2 and MMP-9. We investigated the tissue concentrations of total and active MMP-2 and MMP-9 in AAA walls. Both total and active MMP-2 and MMP-9 showed significantly higher levels in the aortic tissues from patients with AAA than in tissues from control patients (data not shown), which is the same pattern as previous studies have demonstrated. Effect of trapidil on the production of MMP-2 and MMP-9. We subsequently tested whether trapidil had an influence on the protein level of MMP-2 or MMP-9 in AAA tissues. Addition of trapidil (1-100 mol/l) to organ cultures significantly reduced the tissue levels of MMP-2 in a concentration-dependent manner, although it did not alter the tissue concentration of MMP-9 (Fig 1). Suppression of MMP-2 by blocking the CD40-CD40L interaction was also examined by using anti-cd154 antibody. This antibody inhibited the production of MMP-2 (Fig 2), but did not suppress MMP-9 production in cultured AAA walls

4 450 Nagashima et al February 2004 Fig 4. Immunohistochemical analysis. A, Hematoxylin and eosin staining. AAA tissue contains numerous inflammatory cells (magnification, 40 ). B, MMP-2 is positive on macrophages (magnification, 400 ). C and D, MMP-9 is produced by macrophages, a lot of neutrophils circulating in blood vessels, or neutrophils infiltrating into the aortic wall tissue (magnification, 400 ). Fig 5. Immunostaining for CD40. A, Low magnification (40 ). B, High magnification (400 ). CD40 was expressed on B cells, T cells, and macrophages. Neutrophils did not express CD40.

5 Volume 39, Number 2 Nagashima et al 451 Fig 6. Schematic hypothesis for the inhibitory effect of trapidil on AAA formation and progression. (data not shown), indicating that the secretion of MMP-2 (but not MMP-9) was controlled by way of the CD40- CD40L pathway in AAA tissues. Neither TIMP-1 production nor TIMP-2 production was affected by trapidil (data not shown). Effect of trapidil on mrna expression of MMP-2 and MMP-9. We examined the effect of trapidil on mrna expression of MMP-2 and MMP-9 in cultured AAA wall tissues. Semiquantitative RT-PCR demonstrated that trapidil treatment (10 mol/l) suppressed MMP-2 mrna expression but not MMP-9 mrna expression (Fig 3). Immunohistochemical analysis. The walls of AAAs contained many inflammatory cells, such as T cells, B cells, macrophages, mast cells, and neutrophils. Staining for MMP-2 was positive in the macrophages, whereas MMP-9 was positive in the macrophages and a lot of neutrophils (Fig 4), indicating that these cells were the source of MMP-2 and MMP-9 in AAA tissues. After the treatment of trapidil, MMP-2 positive macrophages were not detected (Fig 4), although MMP-9 positive neutrophils could be demonstrated (data not shown). Smooth muscle cells have also been reported to produce MMPs, 9,10 but no smooth muscle cells with positive staining for MMP-2 or MMP-9 were detected in AAA tissues examined in the present study. Previous studies have focused on the interaction between CD40 expressed by macrophages and CD40L expressed by T cells in atherosclerotic lesions. In the present examinations, CD40 was expressed mainly on T cells, B cells, and macrophages but not on neutrophils (Fig 5). CD40-positive vascular smooth muscle cells were not detected. DISCUSSION In atherosclerotic plaques, the importance of the activation of inflammatory cells and subsequent MMP secretion has been emphasized for its instability. 22,23 Aneurysmal degeneration of the aorta also involves destructive remodeling of the aortic wall matrix that is associated with chronic inflammation and local overproduction of MMPs. 4,5 The present study demonstrated two major findings. First, the incubation with trapidil suppressed both the mrna expression and protein production of MMP-2 in cultured AAA tissues. Previous studies have demonstrated up-regulation of MMP-2 expression 6 and MMP-2 activity 9 in AAA. Although a relationship between serum MMP-2 level and the clinical course of patients with AAA has not been determined, a recent study revealed the synergistic role of MMP-2 and MMP-9 in experimental AAA formation in mice, 13 indicating that MMP-2 might play a critical role in the pathogenesis and development of AAA. Trapidil is an antiplatelet agent. Recent studies have demonstrated that trapidil not only has platelet-dependent effects but also possesses platelet-independent effects mediated by way of the CD40-CD40L pathway that involve modulating the expression of various molecules, including MMPs, indicating a potential of trapidil for stabilizing arterial plaques. 17 In fact, the possibility for the superiority of trapidil over aspirin was demonstrated by a multicenter study in patients after acute myocardial infarction, 20 suggesting that trapidil could exert actions beyond its antiplatelet effect clinically. In vitro studies have revealed that doxycycline inhibits activity of MMPs, 24 and this drug has a clinical benefit against AAA progression. 25,26 Compared with doxycycline,

6 452 Nagashima et al February 2004 PCR primers used in this study Gene Primer sequences PCR products, bp MMP-2 sense 5 -AGATCTTCTTCTTCAAGGACCGGTT antisense 5 -GGCTGGTCAGTGGCTTGGGGTA-3 MMP-9 sense 5 -GCGGAGATTGGGAACCAGCTGTA antisense 5 -GACGCGCCTGTGTACACCCACA-3 GAPDH sense 5 -TCGTGGAAGGACTCAT antisense 5 -CAGTGTAGCCCAGGATGCC-3 trapidil does not have any evidence for AAA. However, taking the present results together with an evidence for coronary artery disease, trapidil would be useful, particularly for patients with AAA with coronary artery disease. Second, trapidil did not have a detectable inhibitory effect on mrna expression and protein production of MMP-9 in AAA tissues, which differs from the effect on MMP-2 production. Because anti-cd154 antibody also inhibited MMP-2, but not MMP-9 (data not shown), this difference was considered to be through the differential effect on the CD40-CD40L pathway. 16,17 In the AAA wall, CD40 was expressed mainly on T cells, B cells, and macrophages, but not on neutrophils, and previous studies reported that T cells express CD40L in the arterial wall As we showed in the present study, the cellular source of MMP-9 was macrophages and neutrophils whereas MMP-2 was macrophage derived in our AAA samples, although several reports have shown that neutrophils produce not only MMP-9 but also MMP-2. We, therefore, postulated two possibilities for this difference in the effect of trapidil on MMP-2 and MMP-9. One possibility is that MMP-9 was derived from neutrophils to a greater extent than from macrophages in our AAA samples and that trapidil has fewer effects on neutrophils than on macrophages because neutrophils do not express CD40. Another possibility is that MMP-9 production in human AAA is not dependent on the CD40-CD40L pathway, whereas MMP-2 production is dependent. Further investigations will be necessary to elucidate these possibilities (Fig 6). The CD40-CD40L interaction is a critical step for the initiation of immune response, and this pathway has been reported to be involved in various inflammatory disorders, including atherosclerotic diseases, 18,19 indicating a potential of the CD40-CD40L inhibition therapy for these diseases. Trapidil, a CD40-CD40L pathway inhibitor, is a promising drug for atherosclerotic diseases such as coronary artery disease and/or AAA as shown in the present study. The maximal drug concentration after the administration of clinical dose (100 mg) of trapidil is 16.1 M, which is referred from the Japan Pharmaceutical Information Center. 17 The concentration of trapidil used in our organ culture experiments was close to this level in humans, indicating that the effect on AAA tissues shown in the present ex vivo study might be reproduced in the clinical setting. Limitations. In the present study, mrna expression was evaluated by RT-PCR, and EIA was used to measure MMP protein levels. We were unable to examine the enzymatic activity of MMPs by enzyme zymography because of the limited size of the human samples obtained from patients. MMPs are secreted as a complex with TIMPs by most cells, so it might be reasonable to expect that TIMP-1 or TIMP-2 could also be affected by trapidil. However, we found that the TIMP-1 and TIMP-2 concentrations in AAA tissues were not changed by incubation with trapidil for 48 hours (data not shown). Although the present analysis confirmed that neutrophils secrete MMP-9 in AAA tissues, the contribution of neutrophils in vascular remodeling is controversial. 27,28 Further investigations will be necessary to elucidate the functional role of neutrophils in the pathogenesis of AAA. Conclusions. The present study demonstrated that trapidil, a CD40-CD40L pathway inhibitor, can suppress the mrna expression and protein production of MMP-2 that contributes to elastolysis and remodeling of AAAs, suggesting that trapidil might have a potential clinical value for preventing or treating this disease. We thank Hiroaki Nagao for his technical assistance with the immunohistochemical studies and Katsunori Shimada (Statz Institute Inc.) for the statistical analysis. We also thank Yutaka Kato for scientific discussion and Masahiro Hirabayashi for his generous support and encouragement during this study. REFERENCES 1. Lederle FA, Johnson GR, Wilson SE, Chute EP, Littooy FN, Bandyk D, et al. Prevalence and associations of abdominal aortic aneurysm detected through screening. Aneurysm Detection and Management (ADAM) Veterans Affairs Cooperative Study Group. Ann Intern Med 1997;126: Dardik A, Lin JW, Gordon TA, Williams GM, Perler BA. Results of elective abdominal aortic aneurysm repair in the 1990s: a populationbased analysis of 2335 cases. J Vasc Surg 1999;30: Thompson RW. Basic science of abdominal aortic aneurysms: emerging therapeutic strategies for an unsolved clinical problem. Curr Opin Cardiol 1996;11:

7 Volume 39, Number 2 Nagashima et al Dobrin PB, Mrkvicka R. Failure of elastin or collagen as possible critical connective tissue alterations underlying aneurysmal dilatation. Cardiovasc Surg 1994;2: Shah PK. Inflammation, metalloproteinases, and increased proteolysis: an emerging pathophysiological paradigm in aortic aneurysm. Circulation 1997;96: Davis V, Persidskaia R, Baca-Regen L, Itoh Y, Nagase H, Persidsky Y, et al. Matrix metalloproteinase-2 production and its binding to the matrix are increased in abdominal aortic aneurysms. Arterioscler Thromb Vasc Biol 1998;18: Thompson RW, Holmes DR, Mertens RA, Liao S, Botney MD, Mecham RP, et al. Production and localization of 92-kilodalton gelatinase in abdominal aortic aneurysms. An elastolytic metalloproteinase expressed by aneurysm-infiltrating macrophages. J Clin Invest 1995;96: Curci JA, Liao S, Huffman MD, Shapiro SD, Thompson RW. Expression and localization of macrophage elastase (matrix metalloproteinase- 12) in abdominal aortic aneurysms. J Clin Invest 1998;102: Crowther M, Goodall S, Jones JL, Bell PR, Thompson MM. Increased matrix metalloproteinase-2 expression in vascular smooth muscle cells cultured from abdominal aortic aneurysms. J Vasc Surg 2000;32: Yamashita A, Noma T, Nakazawa A, Saito S, Fujioka K, Zempo N, et al. Enhanced expression of matrix metalloproteinase-9 in abdominal aneurysms. World J Surg 2001;25: Petersen E, Gineitis A, Wagberg F, Angquist KA. Activity of matrix metalloproteinase-2 and -9 in abdominal aortic aneurysms. Eur J Vasc Endovasc Surg 2000;20: McMillan WD, Tamarina NA, Cipollone M, Johnson DA, Parker MA, Pearce WH. Size matters: the relationship between MMP-9 expression and aortic diameter. Circulation 1997;96: Longo GM, Xiong W, Greiner TC, Zhao Y, Fiotti N, Baxter BT. Matrix metalloproteinases 2 and 9 work in concert to produce aortic aneurysms. J Clin Invest 2002;110: Sinha S, Frishman WH. Matrix metalloproteinases and abdominal aortic aneurysms: a potential therapeutic target. J Clin Pharmacol 1998;38: Nagashima H, Aoka Y, Sakomura Y, Sakuta A, Aomi S, Ishizuka N, et al. An HMG-CoA reductase inhibitor, cerivastatin, suppresses production of matrix metalloproteinase-9 in human abdominal aortic aneurysm wall. J Vasc Surg 2002;36: Zhou L, Ismaili J, Stordeur P, Thielemans K, Goldman M, Pradier O. Inhibition of the CD40 pathway of monocyte activation by triazolopyrimidine. Clin Immunol 1999;93: Kato Y, Tsuda T, Hosaka Y, Akahashi T, Shirakawa K, Furusato S, et al. Effects of trapidil on effector functions of monocytes related to atherosclerotic plaque. Eur J Pharmacol 2001;12: Hakkinen T, Karkola K, Yla-Herttuala S. Macrophages, smooth muscle cells, endothelial cells, and T-cells express CD40 and CD40L in fatty streaks and more advanced human atherosclerotic lesions. Colocalization with epitopes of oxidized low-density lipoprotein, scavenger receptor, and CD16 (Fc gammariii). Virchows Arch 2000;437: Schonbeck U, Libby P. CD40 signaling and plaque instability. Circ Res 2001;89: Yasue H, Ogawa H, Tanaka H, Miyazaki S, Hattori R, Saito M, et al. Effects of aspirin and trapidil on cardiovascular events after acute myocardial infarction. Am J Cardiol 1999;83: Nagashima H, Sakomura Y, Aoka Y, Uto K, Kameyama K, Ogawa M, et al. Angiotensin II type 2 receptor mediates vascular smooth muscle cell degeneration in cystic medial degeneration associated with Marfan s syndrome. Circulation 2001;104:I Galis ZS, Sukhova GK, Lark MW, Libby P. Increased expression of matrix metalloproteinases and matrix degrading activity in vulnerable regions of human atherosclerotic plaques. J Clin Invest 1994;94: Libby P. Concepts of the pathogenesis of acute coronary syndromes. Circulation 2001;104: Thompson RW, Baxter BT. MMP inhibition in abdominal aortic aneurysms. Rationale for a prospective randomized clinical trial. Ann N Y Acad Sci 1999;878: Mosorin M, Juvonen J, Biancari F, Satta J, Surcel HM, Leinonen M, et al. Use of doxycycline to decrease the growth rate of abdominal aortic aneurysms: a randomized, double-blind, placebo-controlled pilot study. J Vasc Surg 2001;34: Baxter BT, Pearce WH, Waltke EA, Littooy FN, Hallett JW Jr, Kent KC, et al. Prolonged administration of doxycycline in patients with small asymptomatic abdominal aortic aneurysms: report of a prospective (Phase II) multicenter study. J Vasc Surg 2002;36: Betsuyaku T, Shipley JM, Liu Z, Senior RM. Neutrophil emigration in the lungs, peritoneum, and skin does not require gelatinase B. Am J Respir Cell Mol Biol 1999;20: Takeshita S, Isshiki T, Ochiai M, Ishikawa T, Nishiyama Y, Fusano T, et al. Systemic inflammatory response in acute coronary syndrome: increased activity observed in polymorphonuclear leukocytes but not T lymphocytes. Atherosclerosis 1997;135: Submitted May 10, 2003; accepted Jul 17, 2003.