Membrane fatty acid composition and endothelial cell functional properties.

In order to study the influence of endothelial cell fatty acid composition on various membrane related parameters, several in vitro methods were developed for manipulating the fatty acid content of human endothelial cell membranes. Changes in membrane fatty acid profile were induced by using fatty acid modified lipoproteins or free fatty acids. The largest changes in endothelial fatty acid composition were obtained by culturing the cells in media supplemented with specific free fatty acids. An increase in arachidonic acid content of endothelial phospholipids was induced by supplementation with saturated fatty acids or with arachidonic acid itself. A decrease in arachidonic acid content was obtained by supplementation with other unsaturated fatty acids. Under the experimental conditions used endothelial cells showed a low desaturase activity and a high elongase activity. Considerable alterations in membrane fatty acid composition did not greatly influence certain membrane related parameters such as polymorphonuclear leukocyte adherence and endothelial cell procoagulant activity. In general, for fatty acid modified endothelial cells an association between endogenous arachidonic acid content and total production of eicosanoids was found. This study demonstrates that considerable changes in membrane fatty acid profile affect endothelial cell arachidonic acid metabolism, but it also illustrates homeostasis at the level of endothelial cell functional activity.

Entry of human cytomegalovirus into retinal pigment epithelial and endothelial cells by endocytosis.

PURPOSE: Human retinal pigment epithelial (RPE) cells and endothelial cells (HUVECs) are targets of human cytomegalovirus (HCMV) infection in vivo with significantly protracted replication in vitro compared with that in fibroblasts. This study analyzes the kinetics and mechanisms of HCMV entry into both cell types. METHODS: RPE cells were obtained from donor eyes. HUVECs were isolated from human umbilical cords. HCMV entrance was followed by electron microscopy and immunofluorescence in the presence of lysosomotropic agents and cytochalasin B. RESULTS: Human cytomegalovirus entered into RPE cells and HUVECs as early as 5 minutes after virus- cell contact. Entry was mediated by endocytosis, whereas HCMV enters fibroblasts through fusion. Most internalized viral particles and dense bodies appeared to be degraded within vacuoles. Viral entry, transport of viral proteins to the nucleus, and onset of viral transcription (immediate early [IE] protein expression) were significantly blocked by cytochalasin B. Lysosomotropic agents did not significantly reduce IE expression in RPE cells or HUVECs. CONCLUSIONS: This study shows that HCMV penetrates these highly specialized relevant cells via endocytosis. The low level of infection and the delay in the onset of HCMV expression seen in these cells compared with fibroblasts may be related to the sequestration and degradation of incoming viral particles in endocytic vacuoles.

Intracellular thiol redox status affects rat cytomegalovirus infection of vascular cells.

There is increasing evidence for cytomegalovirus (CMV) induced vascular pathology during acute infection in the immunocompromised host. Inflammation is involved in such processes, which is frequently associated with increased levels of oxidative mediators and reduced anti-oxidant protection. A relation between viral infection and oxidative stress has been recognized for human immunodeficiency virus and herpes simplex virus-1 infections, but little is known in this respect for CMV infections. We investigated if there is a relation between CMV infection of vascular cells and the intracellular redox status using an in vitro rat model. We measured intracellular glutathione levels and rat CMV (RCMV) permissiveness of rat heart endothelial cell lines (RHEC), rat smooth muscle cells (RSMC), and compared these with fully CMV-permissive rat fibroblasts (REF and Rat 2). In addition, the effects of the anti-oxidant N-acetylcysteine (NAC) and the glutathione synthesis inhibitor buthionine sulfoximide (BSO) on CMV permissiveness and replication were investigated in these cell lines. Finally, we investigated infection of vascular cells under inflammatory conditions in an in vivo rat model for acute CMV infection. The results show a very high endogenous glutathione level in RHEC compared to REF, Rat 2 cells and RSMC. This is associated with a low CMV permissiveness in RHEC as opposed to full permissiveness in REF, Rat 2 cells and RSMC in vitro. In addition, modulation of the intracellular thiol redox status affected CMV infection and replication only in RHEC, but not in RSMC and Rat 2 cells. During acute infection in vivo under immunosuppressed conditions rat endothelial cells first become activated and subsequently infected leading to vascular damage and pathology. This study suggests that a high endogenous thiol redox status may contribute to the apparent barrier function of endothelial cells with respect of CMV infection and that oxidative stress may facilitate CMV infection of the vascular wall.

Activation of protein kinase C enhances the infection of endothelial cells by human cytomegalovirus.

The infection of cultured endothelial cells with human cytomegalovirus (HCMV) is generally limited to less than 10% of the cells in contrast to HCMV infection of fibroblasts, where essentially all cells can be infected. It is known that HCMV infection influences a number of signal transduction pathways of infected cells. We therefore questioned whether, conversely, the infectivity of human umbilical vein endothelial cells could be influenced by the deliberate activation of these pathways. When endothelial cells were treated prior to infection with phorbol myristoyl acetate, an activator of protein kinase C, the number of HCMV-positive cells increased two to three times. On the other hand, pretreatment of the cells with RO 31-8220, a specific protein kinase C inhibitor, or with staurosporine, a general protein kinase inhibitor, resulted in a decreased infection level and in abolishment of the PMA-induced effect. Pretreatment with the protein phosphatase inhibitor, okadaic acid, caused a slight increase in infectivity, whereas pretreatment with the protein tyrosine kinase inhibitor, genistein, was without effect. Furthermore, neither forskolin and ilomedine, compounds known to activate the endothelial adenylate cyclase, nor the calcium ionophore A23187 were able to influence HCMV infectivity. It is concluded that: (a) the HCMV infection level of unstimulated endothelial cells is influenced by the basal level of protein kinase C; and (b) stimulation of protein kinase C prior to infection results in an increase of infection by HCMV.

A dual role for endothelial cells in cytomegalovirus infection? A study of cytomegalovirus infection in a series of rat endothelial cell lines.

Several clinical findings point to the involvement of microvascular endothelial cells in cytomegalovirus-related pathology. In this study the interactions of cytomegalovirus (CMV) with microvascular endothelial cells was investigated in an in vitro rat model. A series of rat endothelial cell lines, considered representative for the heterogeneity of heart microvascular endothelium in vivo, were infected with rat CMV (RCMV). The course of infection and production of infectious virus were examined using immunofluorescence staining and plaque titration assays, and was compared with infection of fully permissive rat fibroblasts. These endothelial cell lines displayed differences in susceptibility to CMV infection. Two endothelial cell lines (RHEC 50 and 191) were practically non-permissive, while four endothelial cell lines (RHEC 3, 10, 11 and 116) were partly permissive for CMV infection. In contrast to CMV infection in fibroblasts, only limited infection of the permissive endothelial cell lines was observed without spreading of CMV infection through the monolayer, although infectious virus was produced. Detachment of infected endothelial cells and recovery of the monolayer with time was observed. The detached endothelial cells were able to transmit CMV infection to fibroblast monolayers, but not to endothelial monolayers. Our in vitro results demonstrate differences in permissiveness for RCMV between the series of rat endothelial cell lines, which is suggestive for endothelial heterogeneity to CMV infection in vivo. Our findings indicate that endothelial cells are relatively resistant to CMV infection and that, upon infection, the endothelial monolayer may dispose of the virus via detachment of the infected cells. This points to a dual role for the endothelium in CMV infection in vivo: a barrier for CMV infection (by the endothelial monolayer) on the one hand and spreading of CMV infection (by detached infected cells) on the other hand.

Nuclear import as a barrier to infection of human umbilical vein endothelial cells by human cytomegalovirus strain AD169.

Human embryonal fibroblasts (HEF) are fully permissive for infection by human cytomegalovirus (HCMV) strain AD169, whereas human umbilical vein endothelial cells (HUVEC) seem to form an almost complete barrier to infection with this virus. To investigate this difference in permissiveness, HCMV infection of both cell types was studied using in situ hybridisation (ISH) as well as immunocytochemistry to detect viral DNA and viral proteins. At 2 h post-infection (p.i.), viral DNA was detected dispersed throughout the cytoplasm in both HEF and HUVEC, indicating that HCMV enters all cells of both cell types. At 4 h p.i., the viral DNA was found in the nucleus in HEF, and at the same time expression of immediate early (IE) antigen was found. In contrast, in HUVEC the expression of the IE proteins occurred in a limited number of cells at 8 h p.i., while in most HUVEC an accumulation of viral DNA around the nuclei was observed at this time point. In HUVEC, the nuclear localisation of viral DNA was detected 16 h p.i. in a minority of cells, indicating that transport of HCMV DNA into the nucleus is considerably slower in HUVEC than in HEF. Furthermore, the number of HUVEC containing HCMV DNA decreased about six-fold between 8 and 48 h p.i., indicating that HCMV DNA is either transported into the nucleus or eliminated. Apparently, the lower permissiveness of HUVEC for the HCMV strain AD169 relative to HEF is due to inefficient transport of HCMV DNA into the nuclei of infected HUVEC.

Differential effects of endothelial cell fatty acid modification on the sensitivity of their membrane phospholipids to peroxidation.

In order to study the relationship between the fatty acid (FA) composition of human umbilical vein endothelial cells (HUVEC) and their susceptibility to oxidative stress, we modified their FA composition by long-term culturing in media supplemented with a saturated, monounsaturated, or polyunsaturated FA. Sensitivity of the cellular phospholipids to peroxidation was monitored by measuring conjugated diene formation and decrease of polyunsaturated FAs induced by CuSO4 and H2O2 in liposomes prepared from the respective phospholipid extracts. The extent of phospholipid peroxidation was found to increase with increasing content of polyunsaturated FAs. In addition, the sensitivity of individual polyunsaturated FAs to peroxidation was directly proportional to the number of double bonds present. However, no unequivocal relationship was observed between conjugated diene formation and the phospholipid unsaturation index (an indicator for the combined effect of number of double bonds and polyunsaturated FA contents of the membrane phospholipids). The results suggest that long-term FA modification of endothelial cells differentially alters the sensitivity of their membrane phospholipids to peroxidation: long-term modification with oleic acid may protect against lipid peroxidation, whereas linoleic acid may increase sensitivity to peroxidation. In contrast to what might be expected, long-term modification of endothelial cells with eicosapentaenoic or docosahexaenoic acid does not increase the sensitivity of phospholipids to peroxidation.

Continuous monitoring of lipid peroxidation by measuring conjugated diene formation in an aqueous liposome suspension.

A method is described for the direct and continuous monitoring of lipid peroxidation in an aqueous suspension of sonicated liposomes. By means of ultraviolet difference spectroscopy using tandem cuvettes, the formation of conjugated dienes during liposome peroxidation can be followed. Using this technique, the effect of the fatty acid composition of liposomes on lipid peroxidation can be studied. The results show that both the extent and the time scale of lipid peroxidation are influenced by the fatty acid composition of the phospholipid liposomes. This was confirmed also by other methods, such as measurement of the formation of lipid hydroperoxides and measurement of the decrease in polyunsaturated fatty acids. The advantage of the method described is the direct and continuous monitoring of phospholipid peroxidation in an aqueous environment, without subsampling and extraction of peroxidation products into organic solvents. Using this experimental approach based on difference spectra the contributions from changes in liposome, CuSO4 and H2O2 concentrations are canceled, thus improving sensitivity. The method can be employed for measuring the susceptibility to peroxidation of membrane phospholipids from fatty acid modified endothelial cells.

Cytomegalovirus infection and vessel wall pathology.

This review focuses on information regarding the cytomegalovirus (CMV) in relation to vessel wall pathology including clinical symptoms, pathogenesis and latency. Evidence obtained for involvement of CMV in vascular pathology will be summarized. CMV infection of vascular cells induces cell activation, which leads to expression of adhesion proteins, MHC molecules, cytokine receptors and the production of cytokines and growth factors. Furthermore, CMV infection enhances the inflammatory response and neointima formation in allograft vessels. From such data it can be hypothesized that there is apparently synergy between inflammatory processes and CMV infection. On the one hand, CMV infection leads to cellular activation, on the other hand inflammatory processes contribute to CMV infection. NF-kappa B-dependent activation may form a link between both processes as it would induce CMV replication and host cell activation. Thus, CMV may influence vascular pathology by enhancing the inflammatory process and cellular activation responses.

Polyunsaturated fatty acids and function of platelets and endothelial cells.

Fish oil diets, rich in n-3 polyunsaturated fatty acids, are considered to have an antithrombotic effect. Both platelets and endothelial cells play a crucial role in the regulation of thrombosis and haemostasis. There is substantial evidence that, in these cells, fish oil-derived polyunsaturated fatty acids can replace the n-6 polyunsaturated fatty acid, arachidonic acid, in the membrane phospholipids and can modify those cellular reactions in which the latter fatty acid participates. However, it now appears that dietary n-3 polyunsaturated fatty acids are less potent than, for example, aspirin in modifying the activation properties of these cell systems. This suggests the possible involvement of additional cells or factors in mediating the antithrombotic potential of fish oil fatty acids.

Long-term fatty acid modification of endothelial cells: implications for arachidonic acid distribution in phospholipid classes.

Human umbilical vein endothelial cells were cultured in various fatty acid-modified media until equilibrium conditions were reached (7-8 days). The effects on the fatty acid composition of phospholipid classes and on the metabolism of arachidonic acid (20:4(n-6)) were studied. The results showed that in every phospholipid class large changes in fatty acid composition, including 20:4(n-6) content, were induced by long-term modification with unsaturated as well as saturated fatty acids. However, the mean levels of saturated and unsaturated fatty acids per phospholipid class remained relatively constant, except for cells modified with oleic acid, which showed an increase in monounsaturated fatty acids at the expense of both saturated and polyunsaturated fatty acids. The rate of incorporation of radiolabeled 20:4(n-6) in endothelial lipids was not influenced by long-term fatty acid modification. Cells modified with 20:4(n-6) (having a high 20:4(n-6) content) tended to "store" excess 20:4(n-6) as the elongated product 22:4(n-6) mainly in phosphatidylserine and ethanolamine phospholipids. On the other hand, endothelial cells modified with 20:5(n-3) (having a low 20:4(n-6) content) differed typically from other fatty acid-modified cells by a relatively high level and high incorporation rate of 20:4(n-6) in phosphatidylinositol, with a low extent of elongation. These results indicate extensive homeostatic control of membrane unsaturation in each phospholipid class and economical control of 20:4(n-6) content in all modified endothelial cells, irrespective of a considerable variation of 20:4(n-6) levels in cellular lipids. Moreover, the observed maintenance of a critical level of 20:4(n-6) in phosphatidylinositol, when 20:4(n-6) supply was strongly decreased, may be important for maintaining proper signal transduction upon endothelial cell stimulation.