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Comparative Study
. 2004 Feb;164(2):589-600.
doi: 10.1016/S0002-9440(10)63148-3.

Enhanced cytomegalovirus infection in atherosclerotic human blood vessels

Pamela L Nerheim  1 , Jeffery L Meier, Mohammad A Vasef, Wei-Gen Li, Ling Hu, James B Rice, Daniel Gavrila, Wayne E Richenbacher, Neal L Weintraub
Affiliations
Comparative Study

Enhanced cytomegalovirus infection in atherosclerotic human blood vessels

Pamela L Nerheim et al. Am J Pathol. 2004 Feb.

Abstract

Human cytomegalovirus (CMV) is a possible co-factor in atherogenesis and vascular occlusion, but its ability to actively infect medium and large blood vessels is unclear. A vascular explant model was adapted to investigate CMV infection in human coronary artery, internal mammary artery (IMA), and saphenous vein (SV). Vascular explants were inoculated with CMV Towne or low-passage clinical isolate and examined in situ for CMV cytopathic effect and immediate-early and early antigens, as indicators of active infection. At 5 to 7 days after inoculation, we found that CMV Towne actively infected eight of eight different atherosclerotic blood vessel explants (coronary artery, n = 4; SV and IMA grafts, n = 4), whereas it only infected 2 of 14 nonatherosclerotic blood vessel explants (SV, n = 10; IMA, n = 4) (P = 0.001). The CMV clinical isolate actively infected none of six sets of nonatherosclerotic SV explants at 5 to 7 days after inoculation. The active CMV infections involved adventitial and, less frequently, intimal cells. A small subset of infected cells in atherosclerotic tissue expresses the endothelial cell marker CD31. Smooth muscle cells residing in both atherosclerotic and nonatherosclerotic blood vessels were free of active CMV infections even after all vascular tissue layers were exposed to the virus. In contrast, active CMV Towne infection was evident at 2 days after inoculation in smooth muscle cells and endothelial cells previously isolated from the SV tissues. We conclude that active CMV infection is enhanced in atherosclerotic blood vessels compared to atherosclerosis-free vascular equivalents, and this viral activity is restricted to subpopulations of intimal and adventitial cells.

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Figures

Figure 1
Figure 1
Viability of human SV explants sustained in culture. A and B: Histological examination of extent of neointima on day of procurement (A) and at 5 days in culture (B) assessed by Verhoeff-van Gieson stain for elastic fibers of fixed tissues. C–E: Detection of cell proliferation by immunohistochemical assay for proliferating cell nuclear antigen in neointima and media (C), along luminal surface (D), and in adventitia and vasa vasorum (E) of SV explants at 6 days in culture. C to E are counterstained with hematoxylin. White arrows point to internal elastic lamina; A, L, M, and N denotes adventitia, lumen, media, and neointima, respectively. A to E are representative sections of multiple SVs. Original magnifications: ×100 (A, B); ×200 (D, E); ×400 (C).
Figure 2
Figure 2
Active CMV infection is greatly restricted in human SV explants. A–C: Immunohistochemical assay for CMV IE/early antigen in SV explant at 7 days after inoculation with CMV Towne (107 PFU). A: CMV+ cells are present in patchy distribution in adventitia (arrows), but are absent in or at cut-margin of luminal endothelium, neointima, and media. B: Adventitial CMV+ cells are randomly scattered or located around vaso vasorum. On high-power view (asterisk), many CMV+ cells exhibit cytomegaly and nuclear inclusions (C), which is also evident on H&E staining (not shown). A to C are representative of multiple sections of quadruplicate SV explants, with 38 ± 9 CMV+ adventitial cells per section. Immunohistochemical assay for CMV early antigen only (not shown) yielded 36 ± 11 CMV+ adventitial cells per section. D and E: Immunohistochemical assay for CMV IE/early antigen in SV explant at 7 days after inoculation with clinical CMV isolate in fibroblast lysate (104–5 PFU). CMV antigen is absent in luminal endothelium, neointima, and media (D), but present in lysate debris (arrowhead) at adventitial surface (E). White arrow points to vaso vasorum. Parallel studies of clinical CMV isolate prepared as cell-free virus or in EC lysate (104 PFU) also revealed absence of active CMV infection at 7 days after inoculation (not shown). A to E are counterstained with hematoxylin. Original magnifications: ×100 (A, B, D, E); ×200 (C).
Figure 3
Figure 3
CMV actively infects ECs and SMCs derived from human SVs. Isolated ECs (A) and SMCs (B) were incubated with GFP-expressing CMV Towne at 10 and 1 PFU/cell, respectively, and examined at 2 days after inoculation by fluorescence confocal microscopy. In separate experiments, infected SMCs (1 PFU/cell) were examined for expression of GFP (green fluorescence, C), CMV IE/early antigen (red fluorescence, D), or both GFP and CMV IE/early protein (yellow fluorescence, E). A to E represent results obtained in triplicate; uninfected ECs and SMCs did not emit GFP- or CMV antigen-specific fluorescence.
Figure 4
Figure 4
Active CMV infection in atherosclerotic SV and IMA bypass graft explants. A–C: Atherosclerotic SV bypass graft at 5 days after inoculation with cell-free CMV Towne (107 PFU/ml). H&E staining reveals fibroatheroma and calcification (A), with high-power view showing CMV CPE in adventitial cells (B). C: Immunohistochemical assay for CMV IE/early antigen shows patch of CMV+ cells in adventitia. White arrow points to calcification; black arrows point to CMV+ cells. A to C are representative of multiple sections of duplicate atherosclerotic SV explants. D and E: Atherosclerotic SV bypass graft of another donor at 7 days after inoculation with CMV Towne (107 PFU/ml). Immunohistochemical assay for CMV IE/early antigen reveals presence of CMV+ adventitial cells (D), with high-power view showing CMV+ cell lining lumen of vasa vasorum (E). Arrowhead points to adventitial surface. D and E are representative of multiple sections of quadruplicate atherosclerotic SV explants; 49 ± 19 CMV+ adventitial cells per section. F: Atherosclerotic IMA bypass graft explant at 5 days after inoculation with CMV Towne (107 PFU/ml). Immunohistochemical assay for CMV IE/early antigen reveals patch of CMV+ adventitial cells, 22 ± 6 CMV+ cells per section. Arrowhead points to adventitial surface. F is representative of multiple sections of this explant. A denotes adventitia. C and F are counterstained with fast red; D and E are counterstained with hematoxylin. Original magnifications: ×100 (A); ×200 (C, D, F), ×400 (B, E).
Figure 5
Figure 5
Active CMV infection in atherosclerotic CA explants after inoculation with cell-free CMV Towne (107PFU/ml). A–C: Left anterior descending CA explant at 7 days after inoculation. H&E staining of fibroatheroma and calcification (asterisk; A). Immunohistochemical assay of CMV IE/early antigen in cell at luminal surface (B) and in adventitia cells (C). Arrow in B points to CMV+ cell; arrowhead in C points to vasa vasorum; 2 ± 1 and 133 ± 23 CMV+ intimal and adventitial cells per section, respectively. D: Right CA explant with fibroatheroma examined by immunohistochemical assay for CMV IE/early antigen at 7 days after inoculation. Arrows point to CMV+ cells located near or at the luminal surface. CMV+ intimal and adventitial cells per section, 2 ± 1 and 201 ± 47, respectively. E and F: Atherosclerotic CA explant of another donor in which an intra-CA stent was removed before culture was subjected to immunohistochemical assay for CMV IE/early antigen in adventitia at 5 days after inoculation. Tissue contains 79 ± 12 CMV+ cells per section. E and F are representative of multiple sections of the vessel. A, L, M, and N denote adventitia, lumen, media, and neointima, respectively. B to E are counterstained with hematoxylin. Original magnifications: ×200 (A, C); ×400 (B, D, E).
Figure 6
Figure 6
Identification of cell types in atherosclerotic CA supporting active CMV infection. Removal of intra-CA stent from atherosclerotic CA exposed underlying SMCs in media to viral inoculum. Adjacent sections of intimal aspect of CA explant at 5 days after inoculation with cell-free CMV Towne (107 PFU/ml) were examined by immunohistochemical assay for CMV early antigen (A–C), CD45 (D), CD31 (E and F), and α-SMC actin (G and H). The black arrows point to cells with CMV CPE. The SMC layer in contact with viral inoculum is evident in C and G. Immunohistochemical assay for each antigen was repeated once or twice on multiple sections of the single diseased CA. A to H are counterstained with hematoxylin. Original magnifications: ×40 (G); ×100 (E); ×200 (B-D, F, H); ×400 (A).
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References

    1. Levi M. CMV endothelitis as a factor in the pathogenesis of atherosclerosis. Cardiovasc Res. 2001;50:432–433. - PubMed
    1. Bruggeman CA, Marjorie HJ, Nelissen-Vrancken G. Cytomegalovirus and atherosclerosis. Antiviral Res. 1999;43:191–200. - PubMed
    1. Epstein SE, Speir E, Zhou YF, Guetta E, Leon M, Finkel T. The role of infection in restenosis and atherosclerosis: focus on cytomegalovirus. Lancet. 1996;348:13–17. - PubMed
    1. Nieto FJ, Adam E, Farzadegan H, Melnick JL, Comstock GW, Szklo M. Cohort study of cytomegalovirus infection as a risk factor for carotid intimal-medial thickening, a measure of subclinical atherosclerosis. Circulation. 1996;94:922–927. - PubMed
    1. Zhou YF, Leon MB, Waclawiw MA, Popma JJ, Yu ZX, Finkel T, Epstein SE. Association between prior cytomegalovirus infection and the risk of restenosis after coronary atherectomy. N Engl J Med. 1996;335:624–630. - PubMed

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