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Prior Cytomegalovirus, Chlamydia pneumoniae or Helicobacter pylori infection and the risk of restenosis after percutaneous transluminal coronary angioplasty
International Journal of Cardiology 73 (2000) 165–171 www.elsevier.com / locate / ijcard
Prior Cytomegalovirus, Chlamydia pneumoniae or Helicobacter pylori infection and the risk of restenosis after percutaneous transluminal coronary angioplasty a,
b
c
c
Joerg Carlsson MD *, Sinisa Miketic MD , Joachim Brom PhD , Rolf Ross , Helmut Bachmann PhD c , Ulrich Tebbe MD b a
Department of Cardiology, Kerckhoff-Klinik, Bad Nauheim, Germany Medizinische Klinik II, Klinikum Lippe-Detmold, Detmold, Germany c Novartis Pharma GmbH, Nuernberg, Germany
b
Received 12 March 1999; received in revised form 2 January 2000; accepted 28 January 2000
Abstract We investigated a possible correlation between the serologic status concerning Cytomegalovirus (CMV), Chlamydia pneumoniae (CP) and Helicobacter pylori (HP) and the occurrence of restenosis in patients undergoing percutaneous transluminal coronary angioplasty for symptomatic coronary artery disease. Tests for anti-CMV IgG, anti-Chlamydia pneumoniae IgG and IgA and HP IgG and IgA were performed with an enzyme-linked immunosorbent assay (ELISA). Restenosis was defined as $50% stenosis at follow-up angiography in a vessel with less than 50% stenosis immediately after PTCA. Of 148 patients, 112 (75.7%) were seropositive for CMV, 75 (50.7%) were seropositive for CP and 78 (52.7%) were seropositive for HP. Restenosis occured in 31.8% of patients. CMV seropositivity was established in 74.5% of patients with restenosis versus 76.2% without restenosis (P50.82), CP seropositivity was established in 44.7% of patients with restenosis versus 53.5% without restenosis (P50.32), HP seropositivity was established in 53.2% of patients with restenosis versus 52.5% without restenosis (P50.94). In contrast to some earlier studies CMV or HP seropositivity could not be found to be associated with the risk of restenosis after coronary intervention. An association between the serological status of CP and restenosis could also not be established. 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Angioplasty; Restenosis; Cytomegalovirus; Chlamydia pneumoniae; Helicobacter pylori
1. Introduction Several microorganisms including Cytomegalovirus (CMV), Chlamydia pneumoniae (CP) and Helicobacter pylori (HP) have recently been linked to the pathogenesis of coronary atherosclerosis and restenosis after intervention [1]. Hypotheses *Corresponding author. Tel.: 149-6032-996-2374; fax: 149-6032-9962236. E-mail address: [email protected] (J. Carlsson)
about a possible association between these infections and coronary heart disease are based on a variety of scientific evidence including the presence of CP DNA in atherectomy specimens [2], the associaion between CP seropositivity and the development of coronary heart disease [3] and myocardial infarction [4]. In a study reported in 1996, prior CMV infection could be identified as the only significant predictor of restenosis in 75 patients undergoing directional coronary atherectomy for symptomatic coronary heart disease [5]. Zhou et al. [5] reported a restenosis rate
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J. Carlsson et al. / International Journal of Cardiology 73 (2000) 165 – 171
of 43% in seropositive patients and 8% in seronegative patients. While the mechanisms of restenosis in general are poorly understood a possible link between CMV and restenosis is the p53 tumor-suppressor gene product [5]. This p53 tumor-suppressor gene product inhibits cell proliferation, possibly including smooth muscle cells, within the vascular wall. CMV DNA is present in atheroscleroic lesions and some of the virus early gene products can inhibit the p53 tumorsuppressor gene product, and these effects may enhance proliferation of smooth muscle cells [1,5]. Because chronic infections with HP and CP may lead to higher fibrinogen levels, thrombus formation can also contribute to the process of restenosis in this context [6]. Other investigators found HP seropositivity to be an independent predictor of restenosis in 112 patients undergoing percutaneous transluminal coronary angioplasty PTCA [7]. Since these results concerning CMV resp. HP and restenosis were intriguing we investigated a possible association between these infections and restenosis in a subgroup of patients of the VERAS trial [8]. Under the impression of the current discussion of CP and atherosclerosis we also investigated the hypothesis that prior C. pneumoniae infection is a risk factor for the development of restenosis after PTCA.
2. Materials and methods The VERAS trial was primarily designed to study the effect of octreotide on coronary restenosis after PTCA [8]. Patients with coronary artery disease underwent PTCA and follow-up angiography at 6 months. The minimal luminal diameter and reference diameter before and after PTCA and at the follow-up were measured with a digital quantitative algorithum. Restenosis was defined as $50% stenosis at the follow-up angiography in a vessel with less than 50% stenosis immediately after PTCA. The possible influence of serostatus of the three agents on restenosis was also calculated on the following alternative definitions of restenosis: loss of $50% of the gain in the percentage of vessel diameter stenosis achieved at PTCA and an absolute loss of .0.72 mm of minimal luminal diameter (MLD). The following calculations were performed: relative gain, MLD post-PTCA 2 MLD pre-PTCA / reference diameter; net gain index,
Table 1 Clinical characteristics of the total VERAS population and the subgroup of patients in the present study Characteristic
VERAS (N5274)
Subgroup (N5148)
P-value
Age (years) Male sex Verum medication a Smoker b Diabetes mellitus
57.368.4 231(84.3%) 137(50%) 179(65.3%) 30(10.9%)
57.268.6 125(84.5%) 75(50.7%) 92(62.2%) 18(12.2%)
0.9671 0.8946 0.5173 0.7080
Dilated vessel c RCA LCx LAD
89(33.3%) 47(17.6%) 131(62.2%)
50(33.8%) 24(16.2%) 74(50.0%)
0.9258 0.7193 0.8550
Restenosis d Clinical events e
4(34.1%) 7(17.2%)
47(31.8%) 22(14.9%)
0.6623 0.5441
a
Octreotide (versus placebo). Current or past smoker. c No data available in seven patients of the VERAS total population. d No data available in 57 patients of the VERAS total population. e Death, myocardial infarction, CABG and repeated PTCA. b
MLD follow-up 2MLD pre-PTCA / reference diameter; and relative loss, MLD post-PTCA 2MLD follow-up / reference diameter. Of 217 VERAS patients, 148 were enrolled in our study. These patients were from four of the eight VERAS study centers. The patients were similar to the total VERAS cohort concerning age, sex and angiographic variables (Table 1). Before PTCA blood samples were collected and stored at 2208C. Tests for anti-CMV IgG, anti-Chlamydia pneumoniae IgG and IgA and HP IgG and IgA were performed with an enzyme-linked immunosorbent assay (ELISA) kit (CMV-IgG-ELA Test, Chlamydien IgG rELISA, Chlamydien IgA rELISA, HP-IgG ELISA, HP-IgA ELISA, medac Diagnostika, Hamburg, Germany). The tests were performed according to the manufacturer’s directions and without knowledge of the angiographic findings. Statistical analyses of frequency counts were performed with the chi-square test. Continuous variables were compared by using Student’s t-test for independent samples. Multivariate analysis was performed to determine clinical and angiographic predictors of restenosis. A P value of ,0.05 was considered significant.
3. Results Of the 148 patients, 112 (75.7%) were seropositive
J. Carlsson et al. / International Journal of Cardiology 73 (2000) 165 – 171
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Table 2 Cytomegalovirus (CMV), Chlamydia pneumoniae (CP) and Helicobacter pylori (HP) seropositivity (IgG) in patients with and without restenosis after PTCA
CMV
CP
HP
Restenosis N547 (31.8%)
No restenosis N5101 (68.2%)
seropositive seronegative
35(74.5%) 12(25.5%)
77(76.2%) 24(23.8%)
0.82
seropositive seronegative
21(44.7%) 26(55.3%)
54(53.5%) 47(46.5%)
0.32
seropositive seronegative
25(53.2%) 22(46.8%)
53(52.5%) 48(47.5%)
0.94
for CMV, 75 (50.7%) were seropositive for CP and 78 (52.7%) were seropositive for HP. The results concerning serological status and coronary restenosis at follow-up are shown in Table 2. There was no association between the serological status of prior CMV, CP or HP infection and restenosis. This negative result did not change when two other definitions of restenosis (see above) were used. Also, using relative gain, relative loss and net gain index led to the same negative results. More detailed angiographic data are shown in Tables 3 and 4. The angiographic characteristics in general were not different between seropositive and seronegative patients regarding the three infections tested. However, in CMV seropositive patients the reference diameter and the minimal luminal diameter (MLD) after PTCA of the dilated vessel was slightly lower than in the group of CMV seronegative patients
P-value
(Table 3). In CP positive patients the MLD at the follow-up angiography was significantly greater than in CP negative patients (Table 4). Concerning HP serological status, no significant differences in angiographic data could be observed in the two groups (Table 4). However, there were no differences between the groups with regard to calculated variables relative gain, relative loss and net gain index. In multivariate analysis only post-angioplasty residual stenosis and diabetes were found to be predictive of restenosis (P,0.05).
4. Discussion Several studies within the past few years have examined the hypothesis that an infectious process may contribute to atherosclerosis and restenosis [1–
Table 3 Angiographic data and Cytomegalovirus (CMV) status CMV seropositive (N5112)
CMV seronegative (N536)
P-value
Before PTCA Reference diameter (mm) MLD a (mm) Stenosis (%)
2.9560.60 0.9660.41 67.4612.60
3.1860.76 1.0760.56 66.0612.80
0.0446 0.1363 0.5210
Immediately after PTCA MLD (mm) Stenosis (%) Relative gain (%)
2.0760.48 31.2612.30 38.0620.00
2.2760.60 28.8611.60 38.0619.00
0.0277 0.2524 0.8130
At six months MLD (mm) Stenosis (%) Relative loss (%) Net gain index (%)
1.6660.59 42.4617.30 14.0619.00 24.0623.00
1.7160.56 43.4617.70 18.0621.00 20.0622.00
0.6446 0.7276 0.5594 0.6207
a
MLD5Minimal luminal diameter.
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Table 4 Angiographic data and Chlamydia pneumoniae (CP) status CP seropositive (N575)
CP seronegative (N573)
P-value
Before PTCA Reference diameter (mm) MLD a (mm) Stenosis (%)
3.1260.64 0.9960.43 68.30612.0
2.9360.66 0.9960.42 66.0613.10
0.0529 0.9241 0.2445
Immediately after PTCA MLD (mm) Stenosis (%) Relative gain (%)
2.2060.59 30.50611.9 39.00620.0
2.0660.45 30.50612.5 37.00622.0
0.0728 0.9893 0.8025
At six months MLD (mm) Stenosis (%) Relative loss (%) Net gain index (%)
1.7760.56 41.10616.5 14.00619.0 25.00620.0
1.5960.59 44.1618.10 16.00621.0 20.00619.0
0.0433 0.2721 0.7337 0.4679
a
MLD5Minimal luminal diameter.
7,9–15]. Associations between four infectious agents and coronary heart disease have been reported: CMV, Herpes simplex, CP and HP. Different methodological approaches have been used to establish the link between these agents and atherosclerosis: seroepidemiological studies [3,4,13], studies localizing infectious agents to arterial lesions [2], animal studies and studies using macrolide antibiotics in coronary patients [10–12,15]. Contradictory results have been published in all study categories. In case of the ROXIS (roxithromycin ischemic syndromes) trial, the contradiction was even between the positive preliminary [10] and the negative final publication [11]. It has been argued that a publication bias in favour of positive studies may have led to the current status in the literature concerning infection and atherosclerosis [1,21]. Concerning restenosis after coronary interventions mainly CMV and HP have been studied with contradictory results [5,7,16–20]. A positive and intriguing study [5] prompted us to investigate the question of prior CMV infection and restenosis in patients included in the VERAS trial. The prevalence of CMV seropositivity in our study was not significantly different from that reported in prior studies. We cannot confirm, however, an earlier study that reported CMV seropositivity to be a risk factor for the development of restenosis in a group of only 75 patients [5]. While the mechanisms of restenosis in general are poorly understood, a possible link between CMV and restenosis is the p53
tumor-suppressor gene product [5]. This p53 tumorsuppressor gene product inhibits cell proliferation possibly including smooth muscle cells within the vascular wall. CMV DNA is present in atheroscleroic lesions and some of the virus early gene products can inhibit the p53 tumor-suppressor gene product, and these effects may enhance proliferation of smooth muscle cells [5]. Despite sophisticated theories about CMV infection and restenosis the results of studies are contradictory: Other working groups [16–18,20] with a total of 597 patients including the present data could not find a relationship between CMV seropositivity and restenosis in contrast to Zhou et al. [5] and Blum et al. [19] with 75 and 65 patients, respectively [5,15]. Unfortunately, on the basis of published data it cannot yet be concluded whether there is or is not a relationship between CMV serologic status and restenosis. The differences may in part be attributed to the arbitrarily defined ‘high’ and ‘low’ anti-CMV IgG antibody titers by Blum et al. [15], although the initial report by Zhou et al. did not need such a definition of subgroups to reach significant results [5]. The use of antibody titers is hampered by great temporal titer fluctuations [9] and the fact that there is no correlation between CMV antibody titers and proof of virus antigen and nucleic acid sequences in the arterial wall [22]. Another difference between studies makes direct comparisons difficult. The study of Zhou et al. [5] that found a positive CMV serostatus associated with
J. Carlsson et al. / International Journal of Cardiology 73 (2000) 165 – 171
restenosis used atherectomy, while the other investigations included mostly balloon angioplasty. Only the study of Muhlestein et al. [23] also included 87 patients (34% of their patient population) after coronary stenting. It has been argued that atherectomy may be associated with a higher degree of vessel wall injury and a potentially higher degree of CMV reactivation. The prevalence of CP seropositivity has been reported to be significantly higher in patients with coronary heart disease compared to subjects without coronary heart disease [3,4]. Although there was a comparably high prevalence of CP in our VERAS cohort, the fact that we did not have a control group makes it impossible to draw any conclusions from this concerning CP and atherosclerosis. However, our investigation is to our knowledge the first to report data on CP seropositivity and restenosis. Buffon et al. [7] reported HP seropositivity to be an independent predictor of restenosis in a cohort of 112 patients undergoing coronary angioplasty. HP seropositive patients had a restenosis rate of 57% versus 10% in seronegative patients (P,0.001) [7]. In contrast to that report we could not find evidence for an association between prior HP infection and restenosis (Tables 2 and 5) with a restenosis rate of 53.2% in seropositive patients and 46.8% in seronegative patients (P50.94). It didn’t make a difference whether we used IgG data or IgA data (IgA data not shown). As it is with contradictory data
169
on CMV and restenosis, further research in larger cohorts of patients is needed to resolve these conflicting results concerning HP infection and restenosis. It would have been interesting if the working groups of Blum et al. [19] and Buffon et al. [7] would have found multiple infections to be predictive of restenosis if they had investigated that point as we did. It is unlikely that the question of restenosis and infection will be answered by additional small studies like the already published ones. A standardized set of reagents, protocols and a core testing laboratory should be used for larger and possibly more diverse populations with differing risk factors for atherosclerosis and infections [1,21,23]. Certain limitations of our study need to be addressed. The assay for CP is not specific, it also reacts with C. trachomatis and C. psittaci. However, this seems to be irrelevant since these infections are rare. Maass and Giefers [24] reported serological data on 412 patients with coronary artery disease and 431 population controls using a microimmunofluorescence test that permits a simultaneous reaction to C. pneumoniae, C. trachomatis and C. psittaci. While patients had a higher seroprevalence of CP IgG than controls (91% versus 81%; P,0.001) the IgG seroprevalence for C. trachomatis was ,1% in both groups. No cases of C. psittaci infection were identified. Since our group of patients does not belong to a high-risk population concerning C. trachomatis infections, this methodological limitation
Table 5 Angiographic data and Helicobacter pylori (HP) status HP seropositive (N578)
HP seronegative (N570)
P-value
Before PTCA Reference diameter (mm) MLD a (mm) Stenosis (%)
3.1060.65 1.0560.42 66.4611.10
2.9360.66 0.9260.42 67.8614.10
0.0870 0.0524 0.4890
Immediately after PTCA MLD (mm) Stenosis (%) Relative gain (%)
2.1560.54 30.9611.50 35.0618.00
2.0960.51 30.1613.00 40.0622.00
0.4562 0.6566 0.5880
At six months MLD (mm) Stenosis (%) Relative loss (%) Net gain index (%)
1.6960.56 42.6616.00 15.0616.00 21.0622.00
1.6560.61 42.8618.80 15.0617.00 25.0624.00
0.6552 0.9577 0.9130 0.5653
a
MLD5Minimal luminal diameter.
J. Carlsson et al. / International Journal of Cardiology 73 (2000) 165 – 171
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seems to be irrelevant for the results and conclusions reported here. Another limitation is that we did not look for new infections during the follow-up period. It is, however, highly unlikely that a statistically meaningful number of patients got infected within the 6 months of follow-up. As it is with all serological studies, antibody titres may fluctuate due to repeated reactivations of latent infection and this may possibly lead to other results than long-term average antibody concentrations. This limitation applies to all studies done in this area; however, it does not apply to HP, since HP antibodies lack such fluctuations [9]. Confounding factors for HP infection and coronary artery disease, e.g., low socioeconomic status, play a role in epidemiological studies but seem to be irrelevant in our study [9]. As discussed before, due to the relatively small size of the study population, a relationship between serostatus of the three infectious agents and restenosis cannot definitely be excluded. The differences in some angiographic variables between groups are probably also due to small sample size. However, an influence of serostatus on the nature and distribution of the atherosclerotic process cannot be excluded. Our negative results are supported, however, by the results published by most other authors (Table 6). The relatively small sample size of our study probably also led to the fact that only diabetes and post-angioplasty residual stenosis were found to be predictors of restenosis while other established factors [25] were not. In conclusion, in contrast to some and in accordance to other earlier studies, CMV or HP seropositivity could not be found to be associated with the risk of
restenosis after coronary intervention. An association between the serological status of CP and restenosis could also not be established. The obvious discrepancies of published results based on different patient populations and methods warrant further research.
References [1] Libby P, Egan D, Skarlatos S. Roles of infectious agents in atherosclerosis and restenosis. An assessment of the evidence and need for future research. Circulation 1997;96:4095–103. [2] Muhlestein JB, Hammond EH, Carlquist JF et al. Increased incidence of Chlamydia species within the coronary arteries of patients with symptomatic atherosclerotic versus other forms of cardiovascular disease. J Am Coll Cardiol 1996;27:1555–61. [3] Saikku P, Leinonen M, Tenkanen L et al. Chronic Chlamydia pneumoniae infection as a risk factor for coronary heart disease in the Helsinki Heart Study. Ann Int Med 1992;116:273–8. [4] Miettinen H, Lehto S, Saikku P et al. Association of Chlamydia pneumoniae and acute coronary heart events in non-insulin dependent diabetic and non-diabetic subjects in Finland. Eur Heart J 1996;17:682–8. [5] Zhou YF, Leon MB, Waclawiw MA et al. Association between prior cytomegalovirus infection and the risk of restenosis after coronary atherectomy. N Engl J Med 1996;335:624–30. [6] Torgano G, Cosentini R, Mandelli C et al. Treatment of Helicobacter pylori and Chlamydia pneumoniae infections decreases fibrinogen plasma levels in patients with ischemic heart disease. Circulation 1999;99:1555–9. [7] Buffon A, Liuzzo GM, Caligiuri G et al. Association between prior Helicobacter pylori infection and the risk of restenosis after coronary angioplasty. Circulation 1997;96:I–650, Suppl. I. [8] von Essen R, Ostermaier R, Grube E et al. Effects of octreotide treatment on restenosis after coronary angioplasty. Results of the VERAS Study. Circulation 1997;96:1482–7. [9] Danesh J, Collins R, Peto R. Chronic infections and coronary heart disease: is there a link? Lancet 1997;350:430–6. [10] ROXIS Study Group, Gurfinkel E, Bozovich G, Daroca A, Beck E, Mautner B. Randomised trial of roxithromycin in non-Q-wave coronary syndromes: ROXIS pilot study. Lancet 1997;350:404–7.
Table 6 CMV and restenosis after percutaneous coronary intervention a First author
No. of patients
Seropositive patients (%)
Restenosis in seropositive patients (%)
Restenosis in seronegative patients (%)
P-value
Thomas [17] Muhlestein [16] Blum [19] b Tiran [18] Zhou [5] Present study
82 255 65 112 75 148
77 77 82 73 65 76
49 16 38 n.s. 43 31
63 17 18 n.s. 8 33
NS NS NS NS 0.002 NS
a b
NS5Not significant; n.s.5not stated. See text.
J. Carlsson et al. / International Journal of Cardiology 73 (2000) 165 – 171 [11] Gurfinkel E, Bozovich G, Beck E et al. Treatment with the antibiotic roxithromycin in patients with acute non-Q-wave coronary syndromes: the final report of the ROXIS study. Eur Heart J 1999;20:121–7. [12] Gupta S, Leatham EW, Carrington D, Mendall MA, Kaski JC, Camm AJ. Elevated Chlamydia pneumoniae antibodies, cardiovascular events and azithromycin in male survivors of myocardial infarction. Circulation 1997;96:404–7. [13] McDonagh TA, Woodward M, Morrison CE et al. Helicobacter pylori infection and coronary heart disease in the North Glasgow MONICA population. Eur Heart J 1997;18:1257–60. [14] Koenig W, Rothenbacher D, Hoffmeister A et al. Infection with Helicobacter pylori is not a major independent risk factor for stable coronary heart disease. Lack of a role of cytotoxin-associated protein A-positive strains and absence of a systemic inflammatory response. Circulation 1999;100:2326–31. [15] Anderson JL, Muhlestein JB, Carlquist J et al. Randomized secondary prevention trial of azithromycin in patients with coronary artery disease and serological evidence for Chlamydia pneumoniae infection. The azithromycin in coronary artery disease: elimination of myocardial infection with Chlamydia (ACADEMIC) study. Circulation 1999;99:1540–7. [16] Muhlestein JB, Carlquist JF, Horne BD et al. No association between prior cytomegalovirus infection and the risk of clinical restenosis after percutaneous coronary interventions. Circulation 1997;96:I–650, Suppl. I. [17] Thomas W, Lele S, Adler S, Goudreau E, Cowley M, Vetrovec G. Lack of evidence for a relationship between cytomegalovirus status in coronary angiographic restenosis. Circulation 1997;96:I–650, Suppl. I.
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[18] Tiran A, Tio R, Oostenfeld E et al. Serological response to human cytomegalovirus in patients undergoing percutaneous transluminal coronary angioplasty. Circulation 1996;94:I–711, Suppl. I. [19] Blum A, Giladi M, Weinberg M et al. High anti-cytomegalovirus (CMV) IgG antibody titer is associated with coronary artery disease and may predict post-coronary balloon angioplasty restenosis. Am J Cardiol 1998;81:866–8. [20] Manegold C, Alwazzeh M, Jablonowski H et al. Prior cytomegalovirus infection and the risk of restenosis after percutaneous transluminal coronary balloon angioplasty. Circulation 1999;99:1290–4. [21] Epstein SE, Zhu J. Lack of association of infectious agents with risk of future myocardial infarction and stroke. Definitive evidence disproving the infection / coronary artery disease hypothesis? Circulation 1999;100:1366–8. [22] Chiu B, Viira E, Tucker W et al. Chlamydia pneumoniae, cytomegalovirus and Herpes simplex virus in atherosclerosis of the carotid artery. Circulation 1997;96:2144–8. [23] Bertrand ME, Bauters C. Cytomegalovirus infection and coronary restenosis. Circulation 1999;99:1278–9. [24] Maass M, Gieffers J. Prominent serological response to Chlamydia pneumoniae in cardiovascular disease. Immunol Infect Dis 1996;6:65–70. [25] Anderson HV, Vignale SJ, Benedict CR, Willerson JT. Restenosis after coronary angioplasty. J Interv Cardiol 1993;6:187–202.
Prior Cytomegalovirus, Chlamydia pneumoniae or Helicobacter pylori infection and the risk of restenosis after percutaneous transluminal coronary angioplasty a,
b
c
c
Joerg Carlsson MD *, Sinisa Miketic MD , Joachim Brom PhD , Rolf Ross , Helmut Bachmann PhD c , Ulrich Tebbe MD b a
Department of Cardiology, Kerckhoff-Klinik, Bad Nauheim, Germany Medizinische Klinik II, Klinikum Lippe-Detmold, Detmold, Germany c Novartis Pharma GmbH, Nuernberg, Germany
b
Received 12 March 1999; received in revised form 2 January 2000; accepted 28 January 2000
Abstract We investigated a possible correlation between the serologic status concerning Cytomegalovirus (CMV), Chlamydia pneumoniae (CP) and Helicobacter pylori (HP) and the occurrence of restenosis in patients undergoing percutaneous transluminal coronary angioplasty for symptomatic coronary artery disease. Tests for anti-CMV IgG, anti-Chlamydia pneumoniae IgG and IgA and HP IgG and IgA were performed with an enzyme-linked immunosorbent assay (ELISA). Restenosis was defined as $50% stenosis at follow-up angiography in a vessel with less than 50% stenosis immediately after PTCA. Of 148 patients, 112 (75.7%) were seropositive for CMV, 75 (50.7%) were seropositive for CP and 78 (52.7%) were seropositive for HP. Restenosis occured in 31.8% of patients. CMV seropositivity was established in 74.5% of patients with restenosis versus 76.2% without restenosis (P50.82), CP seropositivity was established in 44.7% of patients with restenosis versus 53.5% without restenosis (P50.32), HP seropositivity was established in 53.2% of patients with restenosis versus 52.5% without restenosis (P50.94). In contrast to some earlier studies CMV or HP seropositivity could not be found to be associated with the risk of restenosis after coronary intervention. An association between the serological status of CP and restenosis could also not be established. 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Angioplasty; Restenosis; Cytomegalovirus; Chlamydia pneumoniae; Helicobacter pylori
1. Introduction Several microorganisms including Cytomegalovirus (CMV), Chlamydia pneumoniae (CP) and Helicobacter pylori (HP) have recently been linked to the pathogenesis of coronary atherosclerosis and restenosis after intervention [1]. Hypotheses *Corresponding author. Tel.: 149-6032-996-2374; fax: 149-6032-9962236. E-mail address: [email protected] (J. Carlsson)
about a possible association between these infections and coronary heart disease are based on a variety of scientific evidence including the presence of CP DNA in atherectomy specimens [2], the associaion between CP seropositivity and the development of coronary heart disease [3] and myocardial infarction [4]. In a study reported in 1996, prior CMV infection could be identified as the only significant predictor of restenosis in 75 patients undergoing directional coronary atherectomy for symptomatic coronary heart disease [5]. Zhou et al. [5] reported a restenosis rate
0167-5273 / 00 / $ – see front matter 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S0167-5273( 00 )00216-3
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J. Carlsson et al. / International Journal of Cardiology 73 (2000) 165 – 171
of 43% in seropositive patients and 8% in seronegative patients. While the mechanisms of restenosis in general are poorly understood a possible link between CMV and restenosis is the p53 tumor-suppressor gene product [5]. This p53 tumor-suppressor gene product inhibits cell proliferation, possibly including smooth muscle cells, within the vascular wall. CMV DNA is present in atheroscleroic lesions and some of the virus early gene products can inhibit the p53 tumorsuppressor gene product, and these effects may enhance proliferation of smooth muscle cells [1,5]. Because chronic infections with HP and CP may lead to higher fibrinogen levels, thrombus formation can also contribute to the process of restenosis in this context [6]. Other investigators found HP seropositivity to be an independent predictor of restenosis in 112 patients undergoing percutaneous transluminal coronary angioplasty PTCA [7]. Since these results concerning CMV resp. HP and restenosis were intriguing we investigated a possible association between these infections and restenosis in a subgroup of patients of the VERAS trial [8]. Under the impression of the current discussion of CP and atherosclerosis we also investigated the hypothesis that prior C. pneumoniae infection is a risk factor for the development of restenosis after PTCA.
2. Materials and methods The VERAS trial was primarily designed to study the effect of octreotide on coronary restenosis after PTCA [8]. Patients with coronary artery disease underwent PTCA and follow-up angiography at 6 months. The minimal luminal diameter and reference diameter before and after PTCA and at the follow-up were measured with a digital quantitative algorithum. Restenosis was defined as $50% stenosis at the follow-up angiography in a vessel with less than 50% stenosis immediately after PTCA. The possible influence of serostatus of the three agents on restenosis was also calculated on the following alternative definitions of restenosis: loss of $50% of the gain in the percentage of vessel diameter stenosis achieved at PTCA and an absolute loss of .0.72 mm of minimal luminal diameter (MLD). The following calculations were performed: relative gain, MLD post-PTCA 2 MLD pre-PTCA / reference diameter; net gain index,
Table 1 Clinical characteristics of the total VERAS population and the subgroup of patients in the present study Characteristic
VERAS (N5274)
Subgroup (N5148)
P-value
Age (years) Male sex Verum medication a Smoker b Diabetes mellitus
57.368.4 231(84.3%) 137(50%) 179(65.3%) 30(10.9%)
57.268.6 125(84.5%) 75(50.7%) 92(62.2%) 18(12.2%)
0.9671 0.8946 0.5173 0.7080
Dilated vessel c RCA LCx LAD
89(33.3%) 47(17.6%) 131(62.2%)
50(33.8%) 24(16.2%) 74(50.0%)
0.9258 0.7193 0.8550
Restenosis d Clinical events e
4(34.1%) 7(17.2%)
47(31.8%) 22(14.9%)
0.6623 0.5441
a
Octreotide (versus placebo). Current or past smoker. c No data available in seven patients of the VERAS total population. d No data available in 57 patients of the VERAS total population. e Death, myocardial infarction, CABG and repeated PTCA. b
MLD follow-up 2MLD pre-PTCA / reference diameter; and relative loss, MLD post-PTCA 2MLD follow-up / reference diameter. Of 217 VERAS patients, 148 were enrolled in our study. These patients were from four of the eight VERAS study centers. The patients were similar to the total VERAS cohort concerning age, sex and angiographic variables (Table 1). Before PTCA blood samples were collected and stored at 2208C. Tests for anti-CMV IgG, anti-Chlamydia pneumoniae IgG and IgA and HP IgG and IgA were performed with an enzyme-linked immunosorbent assay (ELISA) kit (CMV-IgG-ELA Test, Chlamydien IgG rELISA, Chlamydien IgA rELISA, HP-IgG ELISA, HP-IgA ELISA, medac Diagnostika, Hamburg, Germany). The tests were performed according to the manufacturer’s directions and without knowledge of the angiographic findings. Statistical analyses of frequency counts were performed with the chi-square test. Continuous variables were compared by using Student’s t-test for independent samples. Multivariate analysis was performed to determine clinical and angiographic predictors of restenosis. A P value of ,0.05 was considered significant.
3. Results Of the 148 patients, 112 (75.7%) were seropositive
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Table 2 Cytomegalovirus (CMV), Chlamydia pneumoniae (CP) and Helicobacter pylori (HP) seropositivity (IgG) in patients with and without restenosis after PTCA
CMV
CP
HP
Restenosis N547 (31.8%)
No restenosis N5101 (68.2%)
seropositive seronegative
35(74.5%) 12(25.5%)
77(76.2%) 24(23.8%)
0.82
seropositive seronegative
21(44.7%) 26(55.3%)
54(53.5%) 47(46.5%)
0.32
seropositive seronegative
25(53.2%) 22(46.8%)
53(52.5%) 48(47.5%)
0.94
for CMV, 75 (50.7%) were seropositive for CP and 78 (52.7%) were seropositive for HP. The results concerning serological status and coronary restenosis at follow-up are shown in Table 2. There was no association between the serological status of prior CMV, CP or HP infection and restenosis. This negative result did not change when two other definitions of restenosis (see above) were used. Also, using relative gain, relative loss and net gain index led to the same negative results. More detailed angiographic data are shown in Tables 3 and 4. The angiographic characteristics in general were not different between seropositive and seronegative patients regarding the three infections tested. However, in CMV seropositive patients the reference diameter and the minimal luminal diameter (MLD) after PTCA of the dilated vessel was slightly lower than in the group of CMV seronegative patients
P-value
(Table 3). In CP positive patients the MLD at the follow-up angiography was significantly greater than in CP negative patients (Table 4). Concerning HP serological status, no significant differences in angiographic data could be observed in the two groups (Table 4). However, there were no differences between the groups with regard to calculated variables relative gain, relative loss and net gain index. In multivariate analysis only post-angioplasty residual stenosis and diabetes were found to be predictive of restenosis (P,0.05).
4. Discussion Several studies within the past few years have examined the hypothesis that an infectious process may contribute to atherosclerosis and restenosis [1–
Table 3 Angiographic data and Cytomegalovirus (CMV) status CMV seropositive (N5112)
CMV seronegative (N536)
P-value
Before PTCA Reference diameter (mm) MLD a (mm) Stenosis (%)
2.9560.60 0.9660.41 67.4612.60
3.1860.76 1.0760.56 66.0612.80
0.0446 0.1363 0.5210
Immediately after PTCA MLD (mm) Stenosis (%) Relative gain (%)
2.0760.48 31.2612.30 38.0620.00
2.2760.60 28.8611.60 38.0619.00
0.0277 0.2524 0.8130
At six months MLD (mm) Stenosis (%) Relative loss (%) Net gain index (%)
1.6660.59 42.4617.30 14.0619.00 24.0623.00
1.7160.56 43.4617.70 18.0621.00 20.0622.00
0.6446 0.7276 0.5594 0.6207
a
MLD5Minimal luminal diameter.
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Table 4 Angiographic data and Chlamydia pneumoniae (CP) status CP seropositive (N575)
CP seronegative (N573)
P-value
Before PTCA Reference diameter (mm) MLD a (mm) Stenosis (%)
3.1260.64 0.9960.43 68.30612.0
2.9360.66 0.9960.42 66.0613.10
0.0529 0.9241 0.2445
Immediately after PTCA MLD (mm) Stenosis (%) Relative gain (%)
2.2060.59 30.50611.9 39.00620.0
2.0660.45 30.50612.5 37.00622.0
0.0728 0.9893 0.8025
At six months MLD (mm) Stenosis (%) Relative loss (%) Net gain index (%)
1.7760.56 41.10616.5 14.00619.0 25.00620.0
1.5960.59 44.1618.10 16.00621.0 20.00619.0
0.0433 0.2721 0.7337 0.4679
a
MLD5Minimal luminal diameter.
7,9–15]. Associations between four infectious agents and coronary heart disease have been reported: CMV, Herpes simplex, CP and HP. Different methodological approaches have been used to establish the link between these agents and atherosclerosis: seroepidemiological studies [3,4,13], studies localizing infectious agents to arterial lesions [2], animal studies and studies using macrolide antibiotics in coronary patients [10–12,15]. Contradictory results have been published in all study categories. In case of the ROXIS (roxithromycin ischemic syndromes) trial, the contradiction was even between the positive preliminary [10] and the negative final publication [11]. It has been argued that a publication bias in favour of positive studies may have led to the current status in the literature concerning infection and atherosclerosis [1,21]. Concerning restenosis after coronary interventions mainly CMV and HP have been studied with contradictory results [5,7,16–20]. A positive and intriguing study [5] prompted us to investigate the question of prior CMV infection and restenosis in patients included in the VERAS trial. The prevalence of CMV seropositivity in our study was not significantly different from that reported in prior studies. We cannot confirm, however, an earlier study that reported CMV seropositivity to be a risk factor for the development of restenosis in a group of only 75 patients [5]. While the mechanisms of restenosis in general are poorly understood, a possible link between CMV and restenosis is the p53
tumor-suppressor gene product [5]. This p53 tumorsuppressor gene product inhibits cell proliferation possibly including smooth muscle cells within the vascular wall. CMV DNA is present in atheroscleroic lesions and some of the virus early gene products can inhibit the p53 tumor-suppressor gene product, and these effects may enhance proliferation of smooth muscle cells [5]. Despite sophisticated theories about CMV infection and restenosis the results of studies are contradictory: Other working groups [16–18,20] with a total of 597 patients including the present data could not find a relationship between CMV seropositivity and restenosis in contrast to Zhou et al. [5] and Blum et al. [19] with 75 and 65 patients, respectively [5,15]. Unfortunately, on the basis of published data it cannot yet be concluded whether there is or is not a relationship between CMV serologic status and restenosis. The differences may in part be attributed to the arbitrarily defined ‘high’ and ‘low’ anti-CMV IgG antibody titers by Blum et al. [15], although the initial report by Zhou et al. did not need such a definition of subgroups to reach significant results [5]. The use of antibody titers is hampered by great temporal titer fluctuations [9] and the fact that there is no correlation between CMV antibody titers and proof of virus antigen and nucleic acid sequences in the arterial wall [22]. Another difference between studies makes direct comparisons difficult. The study of Zhou et al. [5] that found a positive CMV serostatus associated with
J. Carlsson et al. / International Journal of Cardiology 73 (2000) 165 – 171
restenosis used atherectomy, while the other investigations included mostly balloon angioplasty. Only the study of Muhlestein et al. [23] also included 87 patients (34% of their patient population) after coronary stenting. It has been argued that atherectomy may be associated with a higher degree of vessel wall injury and a potentially higher degree of CMV reactivation. The prevalence of CP seropositivity has been reported to be significantly higher in patients with coronary heart disease compared to subjects without coronary heart disease [3,4]. Although there was a comparably high prevalence of CP in our VERAS cohort, the fact that we did not have a control group makes it impossible to draw any conclusions from this concerning CP and atherosclerosis. However, our investigation is to our knowledge the first to report data on CP seropositivity and restenosis. Buffon et al. [7] reported HP seropositivity to be an independent predictor of restenosis in a cohort of 112 patients undergoing coronary angioplasty. HP seropositive patients had a restenosis rate of 57% versus 10% in seronegative patients (P,0.001) [7]. In contrast to that report we could not find evidence for an association between prior HP infection and restenosis (Tables 2 and 5) with a restenosis rate of 53.2% in seropositive patients and 46.8% in seronegative patients (P50.94). It didn’t make a difference whether we used IgG data or IgA data (IgA data not shown). As it is with contradictory data
169
on CMV and restenosis, further research in larger cohorts of patients is needed to resolve these conflicting results concerning HP infection and restenosis. It would have been interesting if the working groups of Blum et al. [19] and Buffon et al. [7] would have found multiple infections to be predictive of restenosis if they had investigated that point as we did. It is unlikely that the question of restenosis and infection will be answered by additional small studies like the already published ones. A standardized set of reagents, protocols and a core testing laboratory should be used for larger and possibly more diverse populations with differing risk factors for atherosclerosis and infections [1,21,23]. Certain limitations of our study need to be addressed. The assay for CP is not specific, it also reacts with C. trachomatis and C. psittaci. However, this seems to be irrelevant since these infections are rare. Maass and Giefers [24] reported serological data on 412 patients with coronary artery disease and 431 population controls using a microimmunofluorescence test that permits a simultaneous reaction to C. pneumoniae, C. trachomatis and C. psittaci. While patients had a higher seroprevalence of CP IgG than controls (91% versus 81%; P,0.001) the IgG seroprevalence for C. trachomatis was ,1% in both groups. No cases of C. psittaci infection were identified. Since our group of patients does not belong to a high-risk population concerning C. trachomatis infections, this methodological limitation
Table 5 Angiographic data and Helicobacter pylori (HP) status HP seropositive (N578)
HP seronegative (N570)
P-value
Before PTCA Reference diameter (mm) MLD a (mm) Stenosis (%)
3.1060.65 1.0560.42 66.4611.10
2.9360.66 0.9260.42 67.8614.10
0.0870 0.0524 0.4890
Immediately after PTCA MLD (mm) Stenosis (%) Relative gain (%)
2.1560.54 30.9611.50 35.0618.00
2.0960.51 30.1613.00 40.0622.00
0.4562 0.6566 0.5880
At six months MLD (mm) Stenosis (%) Relative loss (%) Net gain index (%)
1.6960.56 42.6616.00 15.0616.00 21.0622.00
1.6560.61 42.8618.80 15.0617.00 25.0624.00
0.6552 0.9577 0.9130 0.5653
a
MLD5Minimal luminal diameter.
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seems to be irrelevant for the results and conclusions reported here. Another limitation is that we did not look for new infections during the follow-up period. It is, however, highly unlikely that a statistically meaningful number of patients got infected within the 6 months of follow-up. As it is with all serological studies, antibody titres may fluctuate due to repeated reactivations of latent infection and this may possibly lead to other results than long-term average antibody concentrations. This limitation applies to all studies done in this area; however, it does not apply to HP, since HP antibodies lack such fluctuations [9]. Confounding factors for HP infection and coronary artery disease, e.g., low socioeconomic status, play a role in epidemiological studies but seem to be irrelevant in our study [9]. As discussed before, due to the relatively small size of the study population, a relationship between serostatus of the three infectious agents and restenosis cannot definitely be excluded. The differences in some angiographic variables between groups are probably also due to small sample size. However, an influence of serostatus on the nature and distribution of the atherosclerotic process cannot be excluded. Our negative results are supported, however, by the results published by most other authors (Table 6). The relatively small sample size of our study probably also led to the fact that only diabetes and post-angioplasty residual stenosis were found to be predictors of restenosis while other established factors [25] were not. In conclusion, in contrast to some and in accordance to other earlier studies, CMV or HP seropositivity could not be found to be associated with the risk of
restenosis after coronary intervention. An association between the serological status of CP and restenosis could also not be established. The obvious discrepancies of published results based on different patient populations and methods warrant further research.
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Table 6 CMV and restenosis after percutaneous coronary intervention a First author
No. of patients
Seropositive patients (%)
Restenosis in seropositive patients (%)
Restenosis in seronegative patients (%)
P-value
Thomas [17] Muhlestein [16] Blum [19] b Tiran [18] Zhou [5] Present study
82 255 65 112 75 148
77 77 82 73 65 76
49 16 38 n.s. 43 31
63 17 18 n.s. 8 33
NS NS NS NS 0.002 NS
a b
NS5Not significant; n.s.5not stated. See text.
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