1 University of Cambridge, Department of Medicine, Addenbrooke's Hospital, Hills Road, Cambridge CB2 2QQ, UK
2 Department of Histopathology, Papworth Hospital, Cambridge, UK
Correspondence
John H. Sinclair
js{at}mole.bio.cam.ac.uk
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ABSTRACT |
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Present address: University of Cambridge, Division of Virology, Addenbrooke's Hospital, Hills Road, Cambridge CB2 2QQ, UK.
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MAIN TEXT |
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Accumulating data suggest that HCMV remains latent in the bone marrow myeloid progenitor cells, which give rise to monocytes, macrophages and dendritic cells. Consistent with this, all these cell types have been shown to carry latent HCMV (Mendelson et al., 1996; Taylor-Wiedeman et al., 1991
) with reactivation occurring only after differentiation of myeloid progenitors into macrophages or dendritic cells (Soderberg-Naucler et al., 1997
, 2001
). However, bone marrow-derived cells also give rise to endothelial cells (EC) (Goodell et al., 1996
); so it is possible that some EC derived from bone marrow progenitors may also be sites of HCMV latency in healthy carriers, and the ability to detect circulating EC harbouring HCMV (Grefte et al., 1993
) could be due to reactivation of HCMV in these cell types.
Consistent with this, analysis of post-mortem tissue from seropositive transplant recipients has shown the presence of HCMV in EC despite the absence of cytopathic effect (Myerson et al., 1984). Also, it has been reported that the origin of EC may dictate the progression of HCMV infection in vivo (Fish et al., 1998
; Jarvis & Nelson, 2002
). In contrast, EC isolated in lung and gastrointestinal tissue of individuals with HCMV disease are productively infected in vivo (Sinzger et al., 1995
), and in vitro infection of different types of EC has been reported to be dependent on the strain of HCMV rather than the vascular bed of origin of EC (Kahl et al., 2000
; Sinzger et al., 2000
).
The possibility that HCMV may also be carried in vascular smooth muscle cells (SMC) has also been raised. For instance there is circumstantial evidence to link HCMV and atherosclerosis, one of the major causes of morbidity in the developed world (Melnick et al., 1993). It has been suggested that HCMV may be a causative agent of atherogenesis (Fabricant et al., 1978
; Melnick et al., 1993
). In a rat model, RCMV promotes smooth muscle proliferation following aortic grafts (Lemstrom et al., 1993
), which may result in restenosis in humans (Zhou et al., 1996
). However, a meta-analysis found that the published epidemiological evidence for an association between HCMV and coronary heart disease was inconclusive (Danesh et al., 1997
).
While there is good evidence that EC arise from the same progenitor as haematopoietic cells, the origin of SMC is not as well defined. A smooth muscle progenitor population has been identified in circulating blood (Shimizu et al., 2001) and, perhaps more intriguingly, embryonic stem cells expressing the vascular endothelial growth factor receptor Flk-1, a protein also expressed by maturing EC (Drake & Fleming, 2000
), can be differentiated into vascular SMC in vitro and in vivo (Yamashita et al., 2000
). These observations suggest that the precursors of EC may also serve as smooth muscle precursors (Sata et al., 2002
). Consequently, the likelihood that EC and SMC may be derived from a population of myeloid progenitors warrants a detailed analysis of whether these cell types may carry latent HCMV in vivo.
Consequently, we have asked specifically whether HCMV DNA can be detected in vascular SMC and EC of the microvasculature in normal, healthy, seropositive individuals under conditions that routinely detect HCMV in CD34+ bone marrow progenitors. To perform our study, SMC and EC were isolated from saphenous vein tissue samples collected from patients who were undergoing cardiovascular surgery, and cultured in vitro. EC were isolated following collagenase digestion of the medial surface of the saphenous tissue and culture in Cs-C medium supplemented with endothelial growth factor and supplement (Sigma). The medial layer was nicked, allowing the upper vascular smooth muscle layer to be peeled off. Segmentation of the tissue and culture in a minimal medium facilitated the outgrowth of morphologically distinct SMC from the edges of the isolated tissue.
To test the identity of the cultured cells, eight-well slides were seeded with either putative EC or SMC, and stained for cell-specific markers or with an IgG isotype-matched control to confirm specificity. All primary antibodies were detected using an appropriate FITC-conjugated secondary antibody (Sigma). Fig. 1(a) shows that, following culture on plates coated with EC attachment factor (Sigma), adherent cells stained positively for endothelium protein (anti-Pal-E; Sera-lab) thus confirming their identity as EC. Similarly, the identity of the SMC was confirmed by staining positively for smooth muscle actin and also by their distinct morphology (Fig. 1c
). The specificity of the staining was confirmed by using isotype-matched controls (Sigma) that showed no staining of the same cells (Fig. 1b and d
).
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Fig. 2(c) shows a similar analysis of DNA isolated from SMC, showing one representative seropositive individual. SMC DNA was amplified in an IE-specific PCR, and again no amplified product could be detected following PCR/Southern blot analysis of 10 subaliquots each containing 1 µg total cellular DNA (lanes 211). Multiple controls showed that DNA from monocytes of a seropositive donor under identical conditions showed IE-specific amplification products (lane 27). Finally, a 300 bp product of the
-globin-specific PCR could be amplified from multiple subaliquots of the SMC DNA (lanes 2830). These observations were representative of multiple SMC DNA samples from each of the 11 seropositive individuals. The data obtained are summarized in Table 1
.
The data presented here show that endogenous HCMV DNA cannot be detected routinely in EC or SMC populations isolated from the saphenous vein of seropositive individuals using a highly sensitive, IE-specific PCR that routinely detects HCMV DNA in myeloid cells, a well established site of HCMV latency. Previous analyses of post-mortem tissue have identified HCMV nucleic acids in the arterial wall tissue of seropositive individuals in the absence of atherosclerosis (Hendrix et al., 1991; Melnick et al., 1994
). However, the exact cell type was undefined, and it is possible that detection of HCMV DNA is the result of contamination with blood cells, such as monocytes, previously shown to carry HCMV genomes (Taylor-Wiedeman et al., 1991
). Also, other studies that have analysed post-mortem tissue suggest that reactivation of HCMV may occur upon death. Thus, the widespread detection of HCMV nucleic acid in a variety of tissues post-mortem may be the result of stress-related virus reactivation rather than detection of latent HCMV genomes (Toorkey & Carrigan, 1989
).
We therefore believe that EC or SMC are unlikely to be a major site of latency of HCMV in normal carriers, despite evidence suggesting that vascular cells could ultimately be derived from myeloid progenitors a progenitor cell type that has been shown to carry latent HCMV in vivo (Mendelson et al., 1996). Although we cannot formally rule out the possibility that the culture conditions used for EC and SMC may lead to a loss of latent viral genomes, we note that HCMV genomic DNA can be detected routinely, even after long-term culture of monocytes from healthy, seropositive individuals (Taylor-Wiedeman et al., 1991
).
We also recognize that the results presented here are from vascular EC and SMC from saphenous vein; aortic EC and SMC (which are much more difficult to obtain ex vivo from human subjects) are not represented in this analysis. However, as vascular EC and SMC appear to originate from the same progenitors as aortic EC and SMC (Sata et al., 2002), it is unlikely that aortic cells will differ substantially from vascular EC and SMC with respect to potential carriage of HCMV in vivo.
Consequently, the failure to detect HCMV DNA in EC and SMC cultured from the saphenous veins of seropositive patients suggests that these cells, as well as aortic cells, are unlikely to represent major sites of HCMV latency in vivo.
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ACKNOWLEDGEMENTS |
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Received 18 May 2004;
accepted 6 July 2004.
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