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(Stroke. 1996;27:1669-1671.)
© 1996 American Heart Association, Inc.

Articles

Human Cytomegalovirus and Restenosis of the Internal Carotid Artery

Paolo Pauletto, MD; Guglielmo Pisoni, MD; Roberta Boschetto, MD; Miranda Zoleo, MD; Achille C. Pessina, MD, PhD Giorgio Palu, MD

the Istituto di Medicina Clinica (P.P., M.Z., A.C.P.) and the Istituto di Microbiologia (G. Pisoni, R.B., G. Palu), Universita di Padova (Italy).

Correspondence to Paolo Pauletto, MD, Associate Professor, Istituto di Medicina Clinica, Universita di Padova, Via Giustiniani, 2, 35126 Padova, Italy. E-mail pauletto@ipdunidix.unipd.it.

	Abstract

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Background Although human cytomegalovirus (HCMV) has been implicated in coronary restenosis, data on the presence of HCMV in the restenosis lesion of the internal carotid artery (ICA) are lacking.

Summary of Report We studied endarterectomy tissue from 5 ICA restenoses and 5 primary atherosclerotic lesions and tissue from 5 normal ICAs. The extracted DNA was tested for HCMV sequences with polymerase chain reaction by use of three primer pairs that amplify different genomic regions. The AD169 strain of HCMV served as the positive control. No trace of the HCMV genome was found in the intima or in the underlying media of endarterectomy specimens from restenosis and primary lesions. The media from control arteries was also HCMV negative.

Conclusions At variance with previous studies carried out in coronary arteries, our results do not support the hypothesis that HCMV infection is implicated in restenosis of the ICA.

Key Words: atherosclerosis • carotid artery diseases • carotid endarterectomy • risk factors

	Introduction

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Despite a large number of studies, the pathophysiological mechanisms underlying the restenosis process are poorly understood.¹ A latent HCMV infection has recently been associated with myointimal proliferation and restenosis of coronary arteries after percutaneous angioplasty.² Sequences from the HCMV IE genes were found in the restenosis tissue. In addition, the HCMV protein IE84, an inhibitor of the growth suppressor protein p53, was highly expressed by SMCs growing from the lesion.² All of this prompted the hypothesis that the occurrence of uncontrolled SMC proliferation is induced by HCMV. However, a subsequent investigation of endarterectomy material from seven patients suffering from coronary restenosis could not detect any HCMV IE gene-specific mRNA.³ In addition, an extensive search in coronary arteries from patients suffering from progressive allograft coronary artery disease failed to show any pathogenetic role for HCMV.⁴

Because data for a role of HCMV in restenosis of carotid arteries are lacking, and the presence of HCMV DNA was only partly tested in the previous reports on coronary arteries, we studied ICA restenosis tissue for the presence of HCMV genome extensively.

We adopted a DNA PCR with a sensitivity of one HCMV genome equivalent per extracted sample. This would rule out the possibility that the low abundance of specific transcripts is responsible for the lack of HCMV detection and hence also explains the above conflicting results.

	Materials and Methods

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Five endarterectomy specimens from patients with ICA restenosis, 5 specimens from patients with primary atherosclerotic lesions, and 5 control ICAs from organ donors were studied. A routine histological examination was carried out with hematoxylin-eosin and Sudan staining.

DNA was extracted from snap-frozen carotid specimens and tested with PCR by use of three sets of HCMV-specific primers. These included the LA-1/LA-6 pair, specific for the pp65 gene (Demmler et al⁵ ), and the UL-1/UL-2 and UL-3/UL-4 pairs, specific for the UL-97 gene (Baldanti et al⁶ ).

To assess reaction competence, PCR was also performed with a primer pair that amplifies a 245-bp fragment of the human ß-globin gene.

Solutions with no DNA and DNA from the AD169 strain of HCMV served as the negative and positive controls, respectively. Both controls were included in each sample.

	Results

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The main characteristics of the patients and of endarterectomy specimens are summarized in the Table. For patients with restenosis, the average time span between first and second revascularization was 16.6±13.2 months.

View this table: [in this window] [in a new window]   Table 1. General Characteristics of Patients and Endarterectomy Specimens

The histological examination showed that in each case the restenosis tissue was mainly fibrous, with scarce lipid content. Fibrous tissue along with Sudan-positive material was found in the primary lesion. Most endarterectomy specimens included some layers of arterial media. The control ICAs were normal, both macroscopically and histologically.

Results of the PCR search for HCMV DNA performed with primers LA-1 and LA-6 are shown in Figs 1 and 2. The analysis revealed a signal corresponding to the ß-globin gene and to the control AD169 DNA. No evidence was obtained for HCMV DNA in samples from restenosis tissue (Fig 1). A similar investigation of endarterectomy specimens from primary atherosclerotic lesions and of normal ICAs failed to detect any HCMV DNA (Fig 2).

View larger version (82K): [in this window] [in a new window]   Figure 1. Testing for HCMV pp65 gene by LA-1 and LA-6. The arrow indicates ß-globin amplification (245 bp). R indicates restenosis; M, molecular weight marker (VIII); AD169, DNA-positive control from the AD169 strain of HCMV; and H2O, water-negative control.

View larger version (102K): [in this window] [in a new window]   Figure 2. Abbreviations are defined in Fig 1. P indicates primary atherosclerotic lesion; C, normal carotid artery.

Similarly, the use of the other primer pairs, UL-1/UL-2 and UL-3/UL-4, did not succeed in highlighting traces of HCMV DNA in any of the examined specimens (not shown).

	Discussion

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The role of HCMV in carotid restenosis has not been investigated thus far. This is probably due to the difficulty of collecting restenosis tissue despite the fact that carotid endarterectomy is followed by a restenosis rate of about 20% (for review, see Reference 1). Cerebral circulation is able to compensate for the chronic hemodynamic deficit, and patients may remain asymptomatic in the presence of severe arterial narrowing or obstruction.

Among the various factors potentially involved in the pathogenesis of coronary restenosis, a latent HCMV infection has been recently emphasized by Speir et al.² These authors reported that 85% of 13 p53-immunopositive restenosis lesions contained HCMV genomic sequences as detected by PCR with use of primers annealing to the IE2 region.² In the same study, none of the test results for 11 primary lesions were positive for IE2 sequences. At variance with this report, the expression of HCMV IE2 genes was not found in a recent investigation that dealt mainly with unstable angina but also included seven cases of coronary restenosis.³ Similar findings were obtained in a different study by Gulizia et al⁴ in samples from 17 patients with allograft coronary artery disease.

In the present work, we concentrated on DNA detection because it can reveal both productive and latent infections. The search for viral RNA may fail during latency since transcription can be either absent or only limited to selected genes, according to the host cell differentiation pattern.⁷ Notwithstanding the high sensitivity of our PCR method, HCMV DNA could not be amplified in the different samples tested. Although a negative result is expected for primary atherosclerotic lesions,² one would anticipate at least some positivity in restenosis specimens and in the normal arterial media.⁸ Since most endarterectomies contained some medial tissue, the presence of HCMV was ruled out, not only from the intima but also from the media. Even if the number of restenosis specimens collected was limited in absolute terms, we can hypothesize that the lack of HCMV detection both in media and intima can be linked to the peculiar SMC composition of the ICA. In fact, different SMC types can be found in the arterial wall according to the developmental stage and the arterial segment considered.⁹ The peculiar differentiative properties of SMCs of the ICA may therefore be associated with some resistance to HCMV infection. On the other hand, HCMV may be carried to the ICA mainly by cell types that do not belong to the stable structure of the arterial wall, such as monocytes,⁷ so that the virus presence in this arterial segment is short-lived and particularly elusive.

Finally, the results of our study, along with data provided by other authors,^{3 4} can be of potential relevance if gene therapy is to be adopted as a new approach to treating vascular disease.¹⁰ Lack of HCMV involvement in atherosclerosis and ICA restenosis may in fact divert attention from IE84/p53 interaction as a candidate target for genetic intervention. Nevertheless, the therapeutic value of endarterial gene transfer to regulate the function of genes controlling SMC proliferation, including p53 itself, is not hampered a priori by the above observations.

This study focuses for the first time on HCMV infection and restenosis of the ICA. It may represent a basis for further investigations particularly aimed at clarifying the existence of an intrinsic resistance of this arterial segment to HCMV infection. These observations would contribute to a better understanding of the nature of the hyperproliferative response of intima.

	Selected Abbreviations and Acronyms

 

HCMV = human cytomegalovirus ICA = internal carotid artery IE = immediate-early PCR = polymerase chain reaction SMC = smooth muscle cell

Received December 27, 1996; revision received May 14, 1996; accepted May 14, 1996.

	References

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1. Pauletto P, Sartore S, Pessina AC. Smooth-muscle-cell proliferation and differentiation in neointima formation and vascular restenosis. Clin Sci. 1994;87:467-479.[Medline] [Order article via Infotrieve]

2. Speir E, Modali R, Huang ES, Leon MB, Shawl F, Finkel T, Epstein SE. Potential role of human cytomegalovirus and p53 interaction in coronary restenosis. Science. 1994;265:391-394.[Abstract/Free Full Text]

3. Kol A, Sperti G, Shani J, Schulhoff N, van der Greef W, Landini MP, La Placa M, Maseri A, Crea F. Cytomegalovirus replication is not a cause of instability in unstable angina. Circulation. 1995;91:1910-1913.[Abstract/Free Full Text]

4. Gulizia JM, Kandolf R, Kendall TJ, Thieszen SL, Wilson JE, Radio SJ, Costanzo MR, Winters GL, Miller LL, McManus BM. Infrequency of cytomegalovirus genome in coronary arteriopathy of human heart allografts. Am J Pathol. 1995;147:461-475.[Abstract]

5. Demmler GJ, Buffone GJ, Schimbor CM, May RA. Detection of cytomegalovirus in urine from newborns by using polymerase chain reaction DNA amplification. J Infect Dis. 1988;158:1177-1184.[Medline] [Order article via Infotrieve]

6. Baldanti F, Silini E, Sarasini A, Talarico CL, Stanat SC, Byron K, Furione M, Bono F, Palu G, Gerna G. A three-nucleotide deletion in the UL97 open reading frame is responsible for the ganciclovir resistance of a human cytomegalovirus clinical isolate. J Virol. 1995;69:796-800.[Abstract]

7. Sinclair J, Sissons JGP. Human cytomegalovirus: pathogenesis and models of latency. Virology. 1994;5:249-258.

8. Hendrix MGR, Dormans PHJ, Kitslaar P, Bosman F, Bruggeman CA. The presence of cytomegalovirus nucleic acids in arterial walls of atherosclerotic and nonatherosclerotic patients. Am J Pathol. 1989;134:1151-1157.[Abstract]

9. Frid MG, Printseva O, Chiavegato A, Faggin E, Scatena M, Koteliansky VE, Pauletto P, Glukhova MA, Sartore S. Myosin heavy-chain isoform composition and distribution in developing and adult human aortic smooth muscle. J Vasc Res. 1993;30:279-292.[Medline] [Order article via Infotrieve]

10. Landau C, Pirwitz MJ, Willard MA, Gerard RD, Meidell RS, Willard JE. Adenoviral mediated gene transfer to atherosclerotic arteries after balloon angioplasty. Am Heart J. 1995;128:1051-1057.

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