CMV induced vascular injury: an electron-microscopic study in the rat.

There is evidence in support of a viral etiology for atherosclerosis. In our study, we demonstrated that cytomegalovirus (CMV) infection in rats caused morphological alterations of the endothelium and subendothelial space of the large vessels and that these alterations are similar to those which are induced by hypercholesterolemia. These alterations consisted of swollen endothelial cells with a surface showing bleb and microvilli formation. In addition, adhesion of leukocytes to the endothelium and the presence of leukocytes (lymphocytes and macrophages) in the subendothelium were found. Lipid accumulation occurred in the endothelium and in the subendothelial space, especially in hypercholesterolemic animals. This lipid accumulation in the subendothelial space consisted of extracellular lipid deposition and of subendothelial located "foam cells". A characteristic phenomenon of the effect of CMV infection of rats was a loosening of the endothelial cells from the basement membrane. The space between the basement membrane and endothelium was expanded and was filed with an increased amount of reticular basal lamina-like material. These observations show that CMV infection is associated with a non-deniding aortic endothelial injury which is consistent with the early events in the process of atherosclerosis.

Cytomegalovirus infection induces vascular injury in the rat.

The role of cytomegalovirus (CMV) in the early development of atherosclerosis was studied in a rat model. Arterial samples derived from virus-infected normo- and hypercholesterolaemic animals were investigated by light microscopy at 1, 4, 8 and 16 weeks post infection. Early atherogenic lesions comparable to those seen in non-infected hypercholesterolaemic rats were found in CMV-infected normocholesterolaemic and hypercholesterolaemic animals, starting at 1 week post infection. The changes consisted of minimal endothelial cell damage, as shown by the en face technique, and a more than 10-fold increase in the number of leukocytes adhering to the aortic intima. The increased adhesion of leukocytes was observed in infected normocholesterolaemic rats but only in the non-infected rats which were hypercholesterolaemic. The infection of hypercholesterolaemic rats did not enhance this effect although it resulted in increased migration of the leukocytes into the subendothelial space. CMV infection of normocholesterolaemic rats induced lipid accumulation in the endothelium. In these animals approximately 1% of the endothelial cells contained lipid at 1 week post infection. In the non-infected hypercholesterol-fed animals 10% of the cells contained lipid. CMV infection in these rats induced an extra increase of the lipid-containing endothelial area. The changes in the CMV infected animals largely corresponded with the intimal injury observed in the hypercholesterolaemic rats. These results support the hypothesis that CMV may be one of the factors involved in atherogenesis.

The effect of virus infection on the adherence of leukocytes or platelets to endothelial cells.

It has been reported that atherosclerotic lesions contain genomic material belonging to members of the herpes family. This suggests that latent viral infection may be one of the atherogenic triggers. In this study we show that early infection of endothelial cell monolayers with Herpes Simplex virus type 1 (HSV-1) or Cytomegalovirus (CMV) results in an increased monocyte (MC) and polymorphonuclear leukocyte (PMN) adherence, but not in an increased platelet adhesion. Further, is demonstrated that MC and PMN respond differently to virus infected endothelial cell monolayers: PMN adhesion to CMV infected cells is approximately 430% of the control adherence, while the MC adherence is increased to 160%. Also, a difference in virus acting is observed: the adherence of MC or PMN to HSV-1 infected endothelial cells is caused by a secreted adherence promoting factor, while the adherence of MC or PMN to CMV infected endothelial cells seems to be a cell-bound phenomenon. In addition, it was demonstrated that the augmentation of MC or PMN adherence to virus infected endothelial cells is sensitive to tunicamycin, suggesting that both virus infections induce the expression of glycoproteins on the endothelial cell membrane, which is responsible for the MC and PMN adhesion. Thus, HSV-1 and CMV infection of endothelium results in an increased adherence of leukocytes which is suggested, irrespective of the precise nature of the mechanism of virus induced atherosclerosis, to be the earliest event associated with endothelium cell damage.

Cytomegalovirus induced PMN adherence in relation to an ELAM-1 antigen present on infected endothelial cell monolayers.

In human umbilical vein endothelial cells infected with cytomegalovirus (CMV), an activation antigen recognized by monoclonal antibody (mAb) ENA1 appeared. mAb ENA1 reacts with an inducible endothelial surface antigen which has characteristics similar to those of ELAM-1. Incubation with anti-IL-1 partly inhibited this appearance and, parallel to this, the virus-induced polymorphonuclear cell (PMN) adhesion was decreased. In addition, the adhesion of PMN to virus-infected endothelial cells could be reduced by F(ab)2 fragments of mAb ENA1 to almost control level. The results obtained after incubation of PMN with mAb IB4 (against CD18) suggest that the adhesion of PMN to uninfected endothelial cells is CD18 glycoprotein dependent, and virus infection up-regulates this glycoprotein-dependent mechanism. These results indicate that the virus-induced PMN adhesion is regulated by the following mechanism: virus infection of endothelial cells induces IL-1 production, and the autocrine IL-1 causes the expression of ELAM-1 on the surface of endothelial cells. In turn this activation antigen ELAM-1 binds with its putative ligand present on the PMN membrane. The virus-induced PMN adhesion occurs also through a CD18 glycoprotein-dependent mechanism.

The effect of cytomegalovirus infection on the adherence of polymorphonuclear leucocytes to endothelial cells.

Adherence of polymorphonuclear leucocytes (PMN) to the endothelial lining of blood vessels is an essential component of the inflammatory response. In this study the effect of cytomegalovirus (CMV) infection on the adherence of PMNs has been examined using an in vitro model system. Human umbilical venous endothelial cells (HUVEC) were grown on fibronectin-coated plastics. CMV infection of HUVEC resulted in the appearance of viral antigens in a small percentage of the cells. At 24 h post-infection when no virus-induced cytopathic effect could be observed in the cell monolayers, the adherence of PMNs was significantly increased. The virus-induced adherence effect was cell bound and could not be induced by soluble components in the medium of the virus-infected cells. The augmentation of the PMN adherence to CMV-infected endothelium was sensitive to tunicamycin suggesting that the virus infection induces the expression of glycoproteins on the HUVEC membranes which are responsible for the PMN adherence. Thus CMV infection of the endothelium results in an increased adherence of PMNs. In the in vivo situation systemic viral infection can potentially lead to infection of blood vessel endothelium and thus can induce a damage of endothelium. This phenomenon could play a role in the atherogenesis process.

Virus induced adherence of monocytes to endothelial cells.

In this study we have demonstrated that infection of human umbilical vein endothelial cells (HUVEC) with Herpes simplex virus type 1 (HSV-1) resulted in an increased adherence of monocytes (MC). This enhanced adherence occurred at 3 h post infection (p.i.) when about 20% of the monolayer is infected and when there is no cytopathic effect observable in the monolayer. The adherence of human MC to virus-infected HUVEC monolayers proved to be effective and reproducible if a multiplicity of infection (MOI) of ten and a ratio of number of MC to number of HUVEC of 5 was used. The increased adherence was also induced by incubating non-infected HUVEC with the 'supernatant medium' of the HSV-1 infected cells, showing that soluble factors induced by viral infection are responsible for the increased adherence. The augmentation of MC adherence to infected endothelium was sensitive to tunicamycin treatment, suggesting that the MC adherence is probably mediated by glycoproteins expressed on the HUVEC membranes by virus infection.