Olufeso, Femi; (2001) The study of cytomegalovirus infection in vascular smooth muscle cells and in an organ culture of the saphenous vein: Potential role in atherogenesis. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Studies in human beings suggest that infectious agents such as cytomegalovirus might be involved in the pathogenesis of vascular diseases such as atherosclerosis, restenosis and transplant-associated atherosclerosis. Smooth muscle cells in the blood vessel wall are involved in the development of these vascular diseases, and CMV infection of these cells may play significant roles in the disease processes. In the present study, an investigation into the potential role of CMV infection of smooth muscle cells in the pathogenesis of atherosclerosis was reported. Smooth muscle cells were isolated and cultured from human saphenous veins and from the veins of the umbilical cord. Aortic smooth muscle cells were also obtained from a commercial source, and served as an arterial alternative to smooth muscle cells isolated from the blood vessel wall of the venous vasculature. Using a high passage CMV strain, AD 169, and a low passage CMV strain, CIF, it was demonstrated that CMV was capable of undergoing a full cycle of replication in smooth muscle cells of venous and arterial origin. Viral replication was demonstrated by the immunofluorescent staining of CMV viral antigens, which are induced following CMV infection of a permissive cell. The replication of CMV in these smooth muscle cell cultures led to the production of infectious virus particles. These studies demonstrated that CMV infection of smooth muscle cells proceeded in a fashion comparable to CMV infection in fully permissive fibroblasts. The changes in the expression of adhesion molecules and MHC antigens following CMV infection of smooth muscle cells were investigated. It was shown that infection with CMV strains AD 169 and CIF significantly increased the constitutive expression of ICAM-1. It was also shown that there was an increase in the expression of LFA-3 molecules on smooth muscle cells following their inoculation with CMV strain AD 169. However the expression of LFA-3 was not altered following the infection of smooth muscle cells with the low passage CMV strain CIF. Increased adhesion molecule expression could potentially increase the infiltration of inflammatory cells, thereby contributing to the development and progression of atherosclerotic lesions. Furthermore, it was demonstrated that CMV potentially interfered with the adaptive immune response, by altering the expression of class I MHC, but not class II MHC molecules on the surface of smooth muscle cells. The infection of smooth muscle cells with CMV strain AD 169 and a low passage CMV strain, CIF, resulted in the down-regulation of class I MHC antigens, suggesting that these cells might be able to evade recognition and killing by cytotoxic T cells and escape immune surveillance, thereby helping CMV to persist in the blood vessel wall. Recently, an in vitro organ culture system of the human saphenous vein has been described where saphenous veins were cultured for extended periods of time in highly serum supplemented media. This system provides the useful advantage over the isolated cell culture technique in that it is possible to maintain the integrity and architecture of the vein wall, and thus the overall effects of CMV infection on the vessel wall can be assessed. Using the organ culture system, whole segments of saphenous veins were cultured in vitro, with the preservation of cell viability and vessel structure. Studies into the infection of these vein segments with CMV strain, AD 169 and strain CIF were undertaken. At various stages post-infection, CMV replication was observed in the neointimal, intimal and adventitial layers of the vessel wall. This was evident by the detection of the expression of CMV-specific IE and early antigens by immunofluorescent staining in these layers of the vein segment. The medial portion of the vessel wall was apparently not infected with CMV. The number of CMV-infected cells in the neointima and adventitia significantly increased with time post-culture, suggesting that the infection was fully permissive, with the resultant spread of the virus in the cells of the vessel wall. CMV infection led to alterations in the structure of the vessel wall, with an increased deposition of extracellular matrix and the detachment of endothelial cells from the basement membrane. The results presented in this thesis provide further support to the premise that CMV infection of vascular smooth muscle cells may play a pathogenic role in the development and progression of atherosclerosis and/or vascular disease. Furthermore, this study has successfully set up important models which could be used in future studies to determine exactly how CMV could contribute to atherogenesis.

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