Function of glutathione peroxidase in endothelial cell vitality.

The two human umbilical vein endothelial cell-derived lines, ECRF24 and ECV304, differ in responsiveness to oxidative stress. In confluent monolayers of ECRF24, but not in ECV304, peroxides induce stress responses such as plasma membrane blebbing and nuclear condensation. The peroxide effect on ECRF24 was preceded by oxidation of reduced glutathione (GSH) and of NAD(P)H, and by oxidation of the redox-sensitive probe, chloromethyl 2',7'-dichlorofluorescin (DCFH). In monolayers of ECV304, peroxides induced only minimal oxidation of GSH, NAD(P)H and DCFH, which was associated with a greatly reduced GSH peroxidase activity in these cells. However, in spite of the absence of a blebbing response, ECV304 were more susceptible than ECRF24 to membrane lipid peroxidation and peroxide-induced necrosis. Only for ECV304, the culturing with high levels of polyunsaturated fatty acids increased lipid peroxidation and cellular death. Treatment of these cells with the GSH peroxidase mimic ebselen effectively reversed their decreased vitality. We conclude that, in peroxide-treated endothelial cells, cell death (necrosis) can result from lipid peroxidation by peroxide that has not been removed by GSH peroxidases, whereas extensive peroxidase activity may cause a stress response (blebbing). The data further identify ECV304 as a stress-sensitive cell line, where peroxides exert their effects independently of GSH oxidation.

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.

Peroxide-induced membrane blebbing in endothelial cells associated with glutathione oxidation but not apoptosis.

Cells under oxidative stress induced by peroxides undergo functional and morphological changes, which often resemble those observed during apoptosis. Peroxides, however, also cause the oxidation of intracellular reduced glutathione (GSH). We investigated the relation between these peroxide-induced effects by using human umbilical vein endothelial cells (HUVEC) and two HUVEC-derived cell lines, ECRF24 and ECV304. With HUVEC, tert-butyl hydroperoxide (tBH) or hydrogen peroxide application in the presence of serum induced, in a dose-dependent way, reorganization of the actin cytoskeleton, membrane blebbing, and nuclear condensation. These processes were accompanied by transient oxidation of GSH. With ECRF24 cells, this treatment resulted in less blebbing and a shorter period of GSH oxidation. However, repeated tBH addition increased the number of blebbing cells and prolonged the period of GSH oxidation. ECV304 cells were even more resistant to peroxide-induced bleb formation and GSH oxidation. Inhibition of glutathione reductase activity potentiated the peroxide-induced blebbing response in HUVEC and ECRF24 cells, but not in ECV304 cells. Neither membrane blebbing nor nuclear condensation in any of these cell types was due to apoptosis, as evidenced by the absence of surface expression of phosphatidylserine or fragmentation of DNA, even after prolonged incubations with tBH, although high tBH concentrations lead to nonapoptotic death. We conclude that, in endothelial cells, peroxide-induced cytoskeletal reorganization and bleb formation correlate with the degree of GSH oxidation but do not represent an early stage of the apoptotic process.

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.

Multiple organ involvement during experimental cytomegalovirus infection is associated with disseminated vascular pathology.

Since much of the pathogenesis of cytomegalovirus (CMV) disease is still unknown and vascular involvement may be of importance a rat model was used to study the nature and course of CMV-induced vascular pathology. In this model, local CMV infection was established by subcutaneous inoculation of rat-specific CMV (RCMV) in the sole of the foot. Sings of endothelial activation, including leucocyte adhesion, preceded detectable RCMV infection of these cells. ultimately, vasculitis and thrombotic occlusion were accompanied by diffuse tissue inflammation and necrosis. Generalized RCMV infection was induced in rats by intraperitoneal administration of the virus, which resulted in multiple organ pathology, including haemorrhages, inflammation, and gastrointestinal ulceration. RCMV-encoded antigens were found especially in mononuclear inflammatory cells in the organs and peripheral blood. In addition, multiple haemorrhages and disturbed haematological parameters indicated diffuse intravascular coagulopathy. In conclusion, this study provides evidence for extensive vascular involvement and haematological consequences during disseminated CMV infection. The nature and chronology of RCM-induced pathological vascular events were demonstrated, indicating the importance of endothelial damage. These data and further study may lead to a better understanding of the pathogenesis of CMV multiple-organ disease.

In vitro tumor angiogenesis assays: plasminogen lysine binding site 1 inhibits in vitro tumor-induced angiogenesis.

It is generally accepted that tumors are angiogenesis-dependent. For research and clinical purposes it would be very attractive to have a simple in vitro model that allows a rapid screening of the angiogenic potential of tumors and to study the effect of angiogenic inhibitors. In vitro angiogenesis models were developed, based on endothelial sprouting/tube formation on a collagen gel, using both tumor cell lines and tumor biopsies. Best results were obtained using conditioned medium of tumor cell lines. In this model it was found that the plasminogen fragment lysine binding site 1 (LBS-1) inhibited in vitro endothelial cell sprouting. This is the first demonstration that LBS-1, which includes angiostatin, is inhibitory for new vessel formation in an in vitro angiogenesis model. We conclude that the assay system allows for rapid and reliable screening of angiogenesis inhibitors.

Cellular arrangement of human breast cancer cell lines determines hemostatic parameters.

Two in vitro models are compared to investigate whether cellular configuration or composition of the matrix in which the cells are cultured influences growth and/or prognostic parameters. T47D, MCF-7 and Hs578T breast cancer cell lines were cultured on two different matrices (agarose and collagen). Growth curves, biological markers (Ki-67, p53 and bcl-2) and the expression of hemostatic parameters were studied. The tested hemostatic parameters were urokinase-type plasminogen activator, tissue-type plasminogen activator and plasminogen activator inhibitor as fibrinolytic parameters and von Willbrand factor, tissue factor, antithrombin III, factor X and factor Xa as coagulation parameters. We found that T47D and MCF-7 formed spheroids in both matrices. Hs578T did not form spheroids; instead, the cells remained single cells in the agarose matrix and grew invasively through the collagen matrix. Expression of the biological markers was similar for spheroids and monolayers. In contrast, a clear difference in expression of hemostatic factors by spheroids and monolayers was found.

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.

Effect of membrane-permeable sulfhydryl reagents and depletion of glutathione on calcium mobilisation in human platelets.

Exposure to peroxides is known to increase the sensitivity of platelets towards activation by agonists. Similar platelet-activating effects are induced by sulfhydryl reagents that evoke Ca2+-induced Ca2+ release (CICR) by stimulating the Ca2+-releasing property of the inositol-1,4,5-trisphosphate receptor. We questioned whether these compounds may act by mobilising intracellular calcium in platelets by altering the intracellular glutathione redox state. Using FURA2-loaded, aspirin-treated platelets, Ca2+ signals were studied following exposure to the membrane-permeable sulfhydryl reagents, thimerosal and disulfiram, the glutathione peroxidase substrate, tert-butyl hydroperoxide, and the inhibitor of glutathione reductase, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). In single platelets monitored by fluorescence imaging techniques, thimerosal and disulfiram elicited repetitive spiking in [Ca2+]i after variable lag times, indicating that these compounds stimulated CICR. BCNU caused [Ca2+]i spiking of only low amplitude, whereas tert-butyl hydroperoxide was inactive. In platelets in suspension devoid of extracellular CaCl2, the sulfhydryl reagents, at concentrations which decreased glutathione by 25%, strongly increased the Ca2+ responses of agonists that stimulated phospholipase C (thrombin) or acted independently of phospholipase C stimulation (thapsigargin). However, Ca2+ release was only slightly promoted by concentrations of BCNU that resulted in substantial depletion of the glutathione level. Tert-butyl hydroperoxide was without effect on glutathione, but partially inhibited Ca2+ mobilisation with these agonists. It is concluded that, in platelets, the potent CICR-promoting effects of sulfhydryl reagents are not solely due to their reaction with intracellular glutathione, but that extensive reduction in glutathione content is associated with Ca2+ mobilisation and CICR.

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.

Tumour angiogenesis: pathophysiology and clinical significance.

In the 1970s it was first proposed that tumours depended on the establishment of a microcirculation in order to grow beyond a few millimetres. Thereafter, the search to prove this hypothesis increased strongly and by the end of the 1980s, evidence was given that tumours were angiogenesis-dependent and metastatic cells were only shed after the tumour had established its microcirculation. The process of neovascularization is regulated by numerous growth factors, vascular endothelial cells, and matrix proteins released from host stromal cells such as macrophages and mast cells. The process of tumour growth and metastasis involves tumour cell-host cell and cell-matrix interactions and many of the underlying mechanisms of these interactions still remain to be elucidated. Although in a minority of cases treatment of solid tumours has been effectively improved, for the majority of cases more adequate treatment is still required to reduce the mortality rate. It has been proposed only recently that specific targeting of the angiogenic process might inhibit tumour growth and metastasis. This promising field of research is now exponentially growing. It is the purpose of this review to summarize current knowledge on the pathophysiology and clinical significance of tumour angiogenesis.

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.

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.

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.

Membrane related effects in endothelial cells induced by human cytomegalovirus.

Previously, we have reported on the increase in procoagulant activity of human umbilical vein endothelial cells (HUVEC) after infection with human cytomegalovirus (HCMV). When using microvascular endothelial cells from foreskin (MVEC), we also observe a significant increase in membrane perturbation and a concomittant increase in procoagulant activity. This effect is both observed with a laboratory HCMV strain (AD169) with low pathogenicity for endothelium and a HUVEC adapted strain (VHL-E) that readily infects endothelial cells. We compared the membrane perturbation of two types of endothelial cells, HUVEC and MVEC with human embryonal fibroblasts (HEF), being fully permissive for both strains. A membrane effect was only found in endothelial cells. Our results suggest that HCMV induces in MVEC more merocyanine-540 incorporation in the membrane as in HUVEC. The increase in the procoagulant activity induced by HCMV was more pronounced in MVEC than in HUVEC. Inactivated virus, as well as virus pre-incubated with heparin was unable to evoke membrane perturbation. It therefore appears that HCMV induces a rapid membrane response in vascular endothelium and that physical interaction of the virion and the endothelial cell is required to elicit this response.

Tumor spheroid-induced vesicle formation on endothelial cells is associated with procoagulant properties.

Fibrin deposits in tumor beds are an intriguing phenomenon. It has been suggested that fibrin plays a role as a provisional matrix in which the tumor grows and induces development of a vascular network. On the other hand fibrin possibly protects the tumor nodule from host defense mechanisms. We therefore investigate whether tumor cells can induce a procoagulant response in endothelial cells leading to fibrin formation. For our studies we employed a modification of the matrix model of Montesano in which sprouting of endothelial cell aggregates can be followed. This system allows us to study in vitro the involvement of coagulation in tumor growth and angiogenesis. Cocultures of endothelial cell aggregates and avascular tumor spheroids in collagen type I gels results in the appearance of extracellular vesicle-like structures on the endothelial sprouts. The vesicles formed on endothelial cell sprouts upon coculturing with tumor cells exhibit an increased amidolytic activity, suggestive of factor X/Xa activity, not dependent on tissue factor exposure. Experiments using HgCl2 and Iodoacetamide point to the importance of SH groups in the factor X/Xa activity on endothelial cell sprouts.

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.

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.