Cytomegalovirus Replication Is Not a Cause of Instability in Unstable Angina
Background Unstable angina is most frequently caused by coronary thrombosis, with or without plaque fissure, but the mechanisms underlying these events are still speculative. Since cytomegalovirus (CMV) antigens and DNA encoding CMV major immediate-early (MIE) gene have been detected in atherosclerotic arterial walls, the active replication of CMV may be responsible for plaque instability. Therefore the expression of CMV MIE gene mRNA, an early marker of viral replication, was assessed in coronary atherectomy specimens from patients with stable or unstable angina.
Methods and Results Twenty patients with unstable angina (12 men and 8 women; mean age, 62 years; range, 44 to 89 years) and 20 patients with stable angina (16 men and 4 women; mean age, 62 years; range, 43 to 81 years) who underwent successful directional coronary atherectomy were enrolled in the study. The efficiency of mRNA extraction, transcription, and amplification from each coronary atherectomy specimen was assessed by performance of reverse transcription and thermal cycling amplification of a 548-bp human β-actin cDNA segment. After Southern blotting and hybridization with a specific probe, all specimens but one showed a positive hybridization signal. The negative sample was excluded from the study. Reverse transcription and thermal cycling amplification of a 145-bp CMV cDNA segment of the MIE gene were then carried out. After Southern blotting and hybridization with a specific probe, none of the specimens showed a positive hybridization signal. Plasmid pACYC 184 containing the Xba I–inserted MIE gene cDNA was used as a positive control: as few as 10 molecules of the plasmid per reaction were detectable after amplification.
Conclusions Our results do not support the hypothesis that, in patients with unstable angina, replication of CMV in coronary atherosclerotic plaques is a major cause of plaque instability. These findings suggest that the research for the causes of unstable angina should be directed toward processes other than CMV replication.
Unstable angina is most frequently precipitated by coronary thrombosis at the site of a previously quiescent atherosclerotic plaque. To date, the causes leading to plaque instability, with or without plaque fissuring,1 are still unknown.2 Recent studies in patients with unstable angina have shown systemic signs of inflammation (elevated C-reactive protein levels3 and lymphocyte activation in blood4 5 ) and histochemical evidence of smooth muscle cell (SMC) proliferation in atherectomy coronary specimens.6 There is also experimental and postmortem evidence that viruses may be involved in the pathogenesis of atherosclerosis.7 In particular, cytomegalovirus (CMV) C87 and AD169 strain-specific antigens8 and DNA encoding CMV major immediate-early (MIE) gene have been detected in atherosclerotic arterial walls.9 10 More recently, an association between CMV infection and the development of restenosis after coronary angioplasty has been suggested.11
In experimental models, viral replication can lead to increased leukocyte adherence to endothelium,12 thrombin generation,13 and stimulation of SMC proliferation.14 For all these reasons it is conceivable that activation of a previously latent CMV infection at the site of an atherosclerotic plaque might favor, at least in some instances, the occurrence of acute coronary syndromes.
The aim of our study was to investigate whether CMV MIE gene mRNA, a marker of early viral replication, is actively expressed in atherectomy coronary specimens from patients with unstable angina compared with patients with stable angina.
Patient Population and Tissue Sampling
Twenty patients with unstable angina15 (12 men and 8 women; mean age, 62 years; range, 44 to 89 years) and 20 patients with stable angina (16 men and 4 women; mean age, 62 years; range, 43 to 81 years) who underwent successful directional coronary atherectomy (residual stenosis <20%) were enrolled in the study. Of the 20 patients with unstable angina, 9 had multi-vessel disease; in these patients, the atherectomy was performed on the coronary branch (or branches) that clinical and angiographic grounds showed to be responsible for the instability.
Coronary specimens (25 from the left anterior descending coronary artery, 8 from the circumflex coronary artery, and 10 from the right coronary artery) were snap-frozen in a denaturing solution (4 mol/L guanidium thiocyanate, 25 mN sodium citrate, pH 7.0, 0.5% sarcosyl, 0.1 mol/L 2-mercaptoethanol)16 in liquid nitrogen and stored at −70°C.
mRNA Reverse Transcription and cDNA Amplification
mRNA was extracted with a QuickPrep Micro mRNA Purification Kit (Pharmacia). To assess the efficiency of the mRNA extraction, reverse transcription was performed according to Sambrook et al17 with Murine Moloney Leukemia Virus Reverse Transcriptase (MuMoLV-RT, Pharmacia) with a specific antisense primer for human β-actin mRNA, which is constitutively expressed in vascular smooth muscle cells. Thermal cycling amplification by Taq polymerase (Pharmacia) of a 548-bp human β-actin cDNA segment was then carried out for 30 cycles. The following conditions were used for each cycle: 1 minute at 95°C to denature the target DNA, 1 minute at 55°C for primer annealing, and 1 minute at 72°C for primer extension. The sequences of the primer set (Genset) complementary to human β-actin cDNA were 5′-GTG-GGG-CGC-CCC-AGG-CAC-CA-3′ (upstream or sense primer) and 5′-CTC-CTT-AAT-GTC-ACG-CAC-GAT-TTC-3′ (downstream or antisense primer, also used for the reverse-transcription reaction).18 Human β-actin cDNA reverse-transcribed from DNase-treated whole blood RNA was used as a positive control; distilled water was used as a negative control.
Thermal cycling amplification of a 145-bp CMV cDNA segment of the MIE gene was carried out for 30 cycles under the same conditions described above. The sequences of the primer set (Genset) complementary to the CMV MIE cDNA segment between positions 2280 and 2425 were 5′-AGC-TGC-ATG-ATG-TGA-GCA-AG-3′ for the upstream or sense primer and 5′-GAA-GGC-TGA-GTT-CTT-GGT-AA-3′ for the downstream or antisense primer.19 The latter was also used for the reverse-transcription reaction. Plasmid pACYC 184 containing the MIE gene cDNA inserted at an Xba I restriction site, kindly provided by Dr M. Stinsky (Department of Microbiology, School of Medicine, University of Iowa), was used as a positive control; distilled water was used as a negative control.
Southern Blotting and Hybridization
The amplification products were run on an agarose (1%) gel according to Sambrook et al20 and transferred (Vacugene System, Pharmacia) onto a nylon membrane (Hybond N, Amersham International). The blot was then hybridized with a specific 32P-γATP–labeled probe in 1× SSC, 0.5% SDS at 45°C. The sequence of the oligomer probe for CMV MIE gene cDNA was 5′-AGG-CCC-GTG-CTA-AAA-AGG-ATG-3′.19 The sequence of the oligomer probe for human β-actin cDNA was 5′-CAT-CGT-CAC-CAA-CTG-GGA-CGA-CAT-3′.18 Autoradiography was performed on a Kodak X-omat S film at −80°C with intensifying screens.
Continuous variables were compared by Student’s t test for unpaired data. Populations were compared by the χ2 test. A value of P≤.05 was considered statistically significant. Results are expressed as mean±1 SD.
The clinical features of the two groups of patients with stable or unstable angina are summarized in the Table⇓. The two groups did not differ significantly in age, sex, number of diseased coronary arteries, and risk factors.
RNA Extraction, cDNA Amplification, and Hybridization
The efficiency of the extraction procedure was confirmed by demonstrating the amplification of a constitutive gene, human β-actin, from the cDNA obtained by reverse transcription of the RNA extracted for each of the atherectomy specimens. All the atherectomy specimens but one showed a positive hybridization signal, after 24-hour exposure, for the 548-bp segment of human β-actin cDNA (Fig 1⇓, top). The negative sample was excluded from further study. To exclude the possibility that the positive signal was due to residual DNA contaminating the extracted mRNA, four samples were treated with DNase (Pharmacia) before reverse transcription. In all cases, a signal similar to that of the untreated samples was seen (data not shown).
CMV MIE Gene mRNA
After 1 week of exposure, CMV MIE gene cDNA amplification product was not detectable in any of the specimens. Conversely, plasmid pACYC 184 containing the Xba I–inserted MIE gene cDNA, used as a positive control, gave a strong hybridization signal (Fig 1⇑, bottom).
To establish the limit of sensitivity of the assay, plasmid pACYC 184, containing the Xba I–inserted MIE gene cDNA, was serially diluted in RNA obtained from tissue specimens. As few as 10 molecules of the plasmid per reaction, corresponding to a concentration of 0.016 fmol/L, were detectable after amplification (Fig 2⇓).
Our hypothesis was that activation of CMV at the site of an atherosclerotic plaque might lead to plaque instability in a sizeable proportion of patients. This was suggested by the observation that CMV infection can lead to increased polymorphonuclear adhesion to endothelium,12 thrombin generation,13 and SMC proliferation,14 which have been observed in unstable angina.2 Our findings do not support this hypothesis, since CMV MIE gene mRNA was not detected in coronary atherectomy specimens obtained from patients with either unstable or stable angina.
CMV is a large DNA virus whose genome codes for several mRNA species that are expressed at different times during viral replication.21 We selected MIE gene mRNA as a target for two main reasons: first, it is expressed early during viral replication22 so that it should be detectable also in case of abortive viral replication and second, even if less abundantly in later stages, this mRNA species is expressed during the whole replicative phase.21 Our study was not designed to evaluate the presence of CMV antigens or DNA. The latter, in the absence of transcripts, would suggest a latent infection,8 9 10 which is unlikely to have the potential to acutely alter plaque stability.
These results are unlikely to be explained by the small size and low cellularity of the sample: in all patients, atherectomy resulted in a residual stenosis of <20%, indicating that the bulk of the atherosclerotic plaque had been removed. Furthermore, human β-actin mRNA, the product of a gene constitutively expressed in SMCs, was efficiently extracted, reverse-transcribed, and amplified from all our specimens but one, demonstrating the adequacy of both the sample size and the experimental procedure. It is also possible that in some of the 9 patients with unstable angina and multivessel disease, the atherectomy was not performed on the culprit lesion. However, the remaining 11 patients had single-vessel disease, and therefore the atherectomy was certainly performed on the culprit lesion.
The value of a negative finding is always related to the sensitivity of the assay used. We determined the sensitivity of our assay: as few as 10 molecules per reaction or 0.016 fmol/L could be detected after amplification. So it can be concluded that, in our atherectomy samples after reverse transcription, the CMV MIE transcripts were either totally absent or present in amounts unlikely to be biologically relevant.
Recently Speir et al11 have suggested a role of CMV in the pathogenesis of restenosis after coronary angioplasty. In such coronary atherectomy specimens they observed a correlation between CMV MIE gene DNA, IE proteins, and the presence of an inactivated form of the p53 protein, which normally has a tumor suppressor function. The number of restenotic lesions in our population (four in unstable angina and three in stable angina) was too small to allow us to draw any conclusion. However, our data are not in disagreement with those reported by Speir and coworkers because they also did not find any sign of CMV infection in primary lesions.
Although our results will have to be confirmed by larger studies, these findings suggest that CMV infection does not play a prevalent role in the pathogenesis of acute coronary syndromes. Other viral infections, such as those caused by herpes simplex or zoster, have the potential to cause local vascular thrombosis.23 There is also evidence of an age-related mitogenic response of vascular SMCs to herpesvirus infection24 and of increased Chlamydia pneumoniae circulating immune-complexes in patients with chronic ischemic coronary heart disease.25 Thus, the failure to identify active replication of CMV in unstable and stable atherosclerotic plaques does not rule out the possibility that viruses or infectious agents other than CMV might play a role in the genesis of acute coronary syndromes.
We are grateful to Dr Felicita Andreotti for critically reading the manuscript and her useful suggestions.
Presented in part at the 67th Scientific Sessions of the American Heart Association, Dallas, Tex, November 14-17, 1994.
- Received December 28, 1994.
- Accepted January 14, 1995.
- Copyright © 1995 by American Heart Association
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