Elevated endothelial microparticle-monocyte complexes induced by multiple sclerosis plasma and the inhibitory effects of interferon-beta 1b on release of endothelial microparticles, formation and transendothelial migration of monocyte-endothelial microparticle complexes

Monocyte migration through the disrupted cerebral endothelial cell (EC) junctions plays an essential role in formation of multiple sclerosis (MS) demyelinating lesions. During pathogenesis of MS, activated ECs release endothelial microparticles (EMP), which possibly facilitate transendothelial migration (TEMIG) of monocytes. To assess functional roles of EMP in MS, specifically, their (i) interaction with monocytes, (ii) effect on monocyte TEMIG in an in vitro model of the brain microvascular endothelial cells (BMVEC), (iii) phenotypic profiles of EMP elicited by MS plasma and (iv) the effects of IFN-beta 1b on release of EMP and on TEMIG of monocytes (mono) and monocytes:EMP complexes (mono:EMP) through the BMVEC. The effect of IFN-beta 1b on the release of EMP and the TEMIG of mono and mono:EMP was assessed by preincubating BMVEC cultures of IFN-beta 1b prior to addition of plasma. Three EMP phenotypes, CD54, CD62E and CD31 were assayed. Plasma specimens from 20 patients with relapsing remitting MS (11 in exacerbation, MS-E, and 9 in remission, ME-R) and 10 healthy controls were studied. Incubation of BMVEC with MS-E plasma yielded elevated levels of EMPCD54, EMP62E and EMPCD31 relative to MS-R and control plasmas. MS-E but not MS-R or control plasma also augmented TEMIG of monocytes, respectively. Mono:EMP complexes further augmented TEMIG relative to mono alone, but only in the presence of MS-E plasma; there was no significant effect with MS-R or control plasmas. The presence of IFN-beta 1b inhibited TEMIG of mono and mono:EMP by 20% and 30%, respectively. MS-E but not MS-R plasma elicited release of activation-derived EMP and enhanced TEMIG of mono and mono:EMP. IFN-beta 1b inhibited TEMIG and release of EMP, suggesting a role of EMP and a novel therapeutic mechanism for IFN-beta 1b in MS.     

Endothelial cells release phenotypically and quantitatively distinct microparticles in activation and apoptosis

BACKGROUND: Endothelial cells (EC) shed endothelial microparticles (EMP) in activation and apoptosis. OBJECTIVES: We compared the antigenic expression of EMP species released during activation as compared to apoptosis, in three cell lines. METHODS: EC from renal and brain microvascular (MiVEC) and coronary macrovascular (MaVEC) origin were incubated with TNF-alpha to induce activation, or deprived of growth factors to induce apoptosis. Antigens expressed on EMP and EC were assayed flow cytometrically and included constitutive markers (CD31, CD51/61, CD105), inducible markers (CD54, CD62E and CD106), and annexin V binding. RESULTS: It was found that in apoptosis, constitutive markers in EMP were markedly increased (CD31>CD105), with a concomitant decrease in expression in EC. Annexin V EC surface binding and annexin V+ EMP were more sharply increased in apoptosis than in activation. In contrast, in activation, inducible markers in EMP were markedly increased in both EMP and EC (CD62E>CD54>CD106). Coronary MaVEC released significantly less EMP than MiVEC. CONCLUSION: EC release qualitatively and quantitatively distinct EMP during activation compared to apoptosis. Analysis of EMP phenotypic signatures may provide clinically useful information on the status of the endothelium.     

Increased percentage of "double phenotype" form T lymphocytes in blood of patients with multiple sclerosis

We studied the percentage of double positive (CD4+CD8+) form of T-cells in a group (total 77) multiple sclerosis (MS) patients, measured by means of monoclonal antibodies anti-CD3, CD4/FITC, CD8/RPE and flow cytometry FACScan (Becton Dickinson). In our study we have shown that the percentage of the double positive T cells is higher (p < 0.05) in the peripheral blood of patients with acute exacerbation of MS (n = 21), and in the course of chronic progressive MS (n = 27) comparing to MS remission (n = 29) and other neurological diseases (n = 25) groups. In the study we have shown that the percentage of double positive T-cells in peripheral blood depends mainly on disease activity.     

Circulating,soluble adhesion proteins in cerebrospinal fluid and serum of patients with multiple sclerosis: Correlation with clinical activity

Soluble adhesion protein intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and endothelial leukocyte adhesion molecule (E-selectin) were measured in serum and cerebrospinal fluid (CSF) of patients with relapsing-remitting multiple sclerosis (RRMS) in remission and in exacerbation, as well as patients with chronic progressive MS, stable MS, and in patients with other neurological and inflammatory diseases (ONDs). Serum ICAM-1 and E-selectin were significantly elevated in patients with MS over those with ONDs and controls. CSF VCAM-1 and E-selectin were found to be elevated over control and disease control samples. No increase in CSF ICAM-1 was observed. Results were analyzed longitudinally and by MS category. In paired CSF and serum samples from patients in exacerbation, elevated VCAM-1 correlated with increased serum VCAM-1 in 5 of 7 patients. Elevated CSF E-selectin did not correlate with elevations in serum E-selectin.     

Endothelial microparticles: a potential contribution to the thrombotic complications of the antiphospholipid syndrome

The antiphospholipid syndrome (APS) refers to persistent anti-phospholipid antibodies (aPL) associated with thrombotic and/or obstetrical complications. The endothelial cell is a target of aPL which can induce a procoagulant and proinflammatory endothelial phenotype, as reported both in vivo and in vitro. Microparticle production is a hallmark of cell activation. In the present study, the presence of endothelial microparticles (EMP) in the plasma of APS patients was investigated. To determine if there is a correlation with certain biological and clinical features, EMP levels were measured in thrombosis-free patients with systemic lupus erythematosus (SLE) patients, with and without aPL, in patients with non aPL-related thrombosis, as well as in healthy controls. Compared to healthy subjects, elevated plasma levels of EMP were found in patients with APS and in SLE patients with aPL, but not in SLE patients without aPL or in non aPL-related thrombosis. EMP levels were also associated with Lupus Anticoagulant (LA) detected by a positive Dilute Russell's Viper Venom time (DRVVT). In parallel, we analyzed the capacity of these plasma to induce vesiculation of cultured endothelial cells. We demonstrated an increase of EMP generated in response to plasma from patients with auto-immune diseases. Interestingly, only APS plasma induced the release of EMP with procoagulant activity. These ex vivo and in vitro observations indicate that generation of EMP in APS and SLE patients results from an autoimmune process involving aPL. Production of procoagulant microparticles in APS patients may represent a new pathogenic mechanism for the thrombotic complications of this disease.     

Soluble and cell surface ICAM-1 as markers for disease activity in multiple sclerosis

Objective - The intercellular adhesion molecule-1 (ICAM-1) is a member of the Ig supergene family. ICAM-1 is expressed on various cells like peripheral blood lymphocytes, endothelial cells or thymic cells and the cell surface form is supposed to be shed into a soluble form. The expression of ICAM-1 is induced by cytokines like Interleukin-1, TNF alpha or interferon gamma. The aim of the study was to investigate whether changes of cell surface and soluble ICAM-1 in the cerebrospinal fluid (CSF) and blood are indicative for disease activity in patients with multiple sclerosis (MS). Material and methods - In all patients with relapsing-remitting MS (relapse: n =31, remission: n = 11) and controls ( n = 13) the expression of cell surface ICAM-1 (c-ICAM-1) was determined by two colour flow cytometry. Soluble ICAM-1 (s-ICAM-1) was measured by ELISA. Follow-up examinations were done 3 months later. Results - In 31 patients with a current relapse we found significantly decreased expression levels of c-ICAM-1 on leukocytes in CSF ( P <0.001) and blood ( P <0.10), when compared to those 11 individuals experiencing remission. In contrast we observed significantly ( P <0.05) increased levels of s-ICAM-1 in CSF of patients with relapses. Comparing patients who had been in remission for more than 4 weeks ( n = ll) with remission lasting longer than 3 months ( n =28) we detected stable c-ICAM-1 expression on CD3 ⁺ T cells in blood. Conclusion - Our results demonstrate for the first time that c-ICAM-1 on CD3 ⁺ T-cells in CSF and blood is an activity marker in MS.     

Is there a link between CD146, a novel adhesion molecule and other markers of endothelial dysfunction in nephrotic syndrome and continuous ambulatory peritoneal dialysis?

BACKGROUND: CD146 is a novel cell adhesion molecule localized at the endothelial junction. Its increased plasma levels in chronic renal failure are linked to endothelial dysfunction. Endothelial dysfunction and hemostatic disturbances, a common feature of nephrotic syndrome (NS), mimics a state of protein loosing by peritoneal membrane in patients on chronic ambulatory peritoneal dialyses (CAPD). The aim of the study was to assess CD146 in relation to other markers of endothelial cell injury in patients with NS in comparison to patients on CAPD. MATERIALS AND METHODS: We studied 45 CAPD patients, 43 patients with nephrotic syndrome and 25 healthy volunteers. Markers of endothelial cell injury: TFPI total, full length, truncated, von Willebrand factor, trombomodulin, P-selectin, E-selectin, ICAM, VCAM and CD146 were assessed using commercially available kits. RESULTS: All these markers studied except selectins were significantly elevated in patients with NS and CAPD when compared to the healthy volunteers. In CAPD, VCAM, thrombomodulin and CD146 were significantly elevated over NS patients. CD146 correlated significantly with ICAM as well as total and truncated TFPI in CAPD patients. Moreover, total TFPI was positively related to VCAM. CD146 correlated with ICAM in NS, whereas in healthy volunteers CD146 correlated only with TFPI concentration. CONCLUSIONS: Our studies indicate that in nephrotic patients, as well as in CAPD, there is an evidence of endothelial cell injury. Correlations between CD146 and adhesion molecules and TFPI might further support its use as a endothelial cell function marker.     

Flow cytometric detection of endothelial microparticles (EMP): Effects of centrifugation and storage alter with the phenotype studied

Introduction

Endothelial microparticles (EMP) are released into the circulation in case of endothelial disturbance, and are therefore increasingly investigated as a biomarker reflecting disease activity. Numerous pre-analytic methods have been proposed for their flow cytometric enumeration, but standardization is still lacking. In this study we evaluated the influence of centrifugation and storage conditions on EMP quantification.

Materials and Methods

Platelet-poor plasma (PPP) from 10 healthy volunteers was prepared by centrifugation at 1 550 g for 20 minutes twice. A first aliquot of PPP was analyzed immediately, a second after storage at 4 °C for 7 hours. A third and fourth aliquot were snap-frozen and stored at -80 °C for 7 and 28 days. A final aliquot was further centrifuged at 10 000 g for 10 minutes and analyzed immediately. EMP were defined as CD31+CD42b-, CD62E+, CD144+ or CD144+CD105+ particles, smaller than 1.0 µm.

Results

High speed centrifugation led to a significant loss of CD31+CD42b- EMP (p = 0.004). A good correlation between PPP and high speed centrifuged PPP was only found for CD144+ EMP (Kendall tau b = 0.611, p = 0.025).Storage at 4 °C did not affect EMP quantification. However, freezing at -80 °C increased CD31+CD42b- and CD62E+ EMP counts, and lowered CD144+ EMP (p < 0.05). Nevertheless, the agreement among the different storage conditions was relatively good (Kendall coefficient of concordance > 0.487; p < 0.05).

Conclusion

The flow cytometric detection of EMP varies with the centrifugation protocol and the storage method used, and these changes also depend on the phenotype studied. The results of this study caution against comparing study results gathered with different EMP laboratory protocols.     

sICAM-1 and TNF-alpha induce MIP-2 with distinct kinetics in astrocytes and brain microvascular endothelial cells

The dysfunction of the blood-brain barrier (BBB) occurring after traumatic brain injury (TBI) is mediated by intracerebral neutrophil accumulation, chemokine release (e.g., interleukin (IL)-8) and upregulation of adhesion molecules (e.g., intercellular adhesion molecule (ICAM)-1). In patients with severe TBI, we previously found that elevated cerebrospinal fluid (CSF) IL-8 and soluble (s)ICAM-1 correlate with BBB dysfunction, and this prompted us to concomitantly monitor IL-8, sICAM-1 and their stimulator tumor necrosis factor (TNF)-alpha in CSF. Potential mechanisms for upregulation of the IL-8 analogue, murine macrophage inflammatory protein (MIP)-2, and sICAM-1 at the BBB were studied using cultured mouse astrocytes and brain microvascular endothelial cells (MVEC). In CSF of seven patients, IL-8 and sICAM-1 were elevated for 19 days after severe TBI, whereas TNF-alpha exceeded normal values on 9 days. Stimulation of MVEC and astrocytes with TNF-alpha simultaneously induced the release of MIP-2 reaching saturation by 4-8 hr and of sICAM-1 increasing continuously from 2-4 hr to 12 hr. Augmented sICAM-1 production correlated with enhanced membrane-bound (m)ICAM-1 expression in both cell types (r(s) = 0.96 and 0.90, P < 0.0001), but was markedly higher in astrocytes. The release of sICAM-1 was not influenced by IL-8 or MIP-2, although astrocytes and MVEC expressed the IL-8/MIP-2 receptor (CXCR-2) as determined by FACS analysis. Instead, we found that sICAM-1 strongly induced MIP-2 secretion by both cell types with kinetics differing from those evoked by TNF-alpha. If added together, sICAM-1 and TNF-alpha synergistically induced MIP-2 production suggesting the involvement of two different pathways for MIP-2 regulation.     

T-cell phenotypic profiles in the cerebrospinal fluid and peripheral blood of multiple sclerosis patients.

Thirty-nine patients with clinically definite multiple sclerosis (MS) entered the study. Of 28 subjects with a relapsing-remitting course, 19 were classified in acute relapse, 9 in remission; 11 patients had a progressive course without remissions. Furthermore, 6 subjects with inflammatory neurological disease (IND), and 10 with non-inflammatory and non-neoplastic neurological disease (NIND) were investigated. We simultaneously studied cerebrospinal fluid (CSF) and peripheral blood (PB) T-, B- and NK-cell subsets, as defined by following monoclonal antibodies: anti-CD3, -CD4, -CD8, -CD19, -CD16, -HLA-DR and -IL-2-R. We found a significant increase of CD4+ T-cells compared with controls in CSF, with respect to PB, of MS patients, particularly in acute relapse. An increase of HLA-DR+ cell percentages in the CSF than in the PB in all MS groups, especially in attacks of MS but also in remission, was also observed, with a positive correlation between CD4+ T-cell and DR+ cell percentages both in the CSF as well as in the PB of relapsing MS patients. These findings, together with the increase of IL-2-R+ cells in the PB, particularly in relapsing MS, give further support for the presence of a systemic T-cell activation in MS.     

Apoptosis of T cells in peripheral blood and cerebrospinal fluid is associated with disease activity of multiple sclerosis

Apoptotic elimination of pathogenic T cells is considered to be one of regulatory mechanisms in multiple sclerosis (MS). To explore the potential relationship between Fas-mediated apoptosis and the disease course of MS, we examined apoptosis, defined by annexin V (AV) binding, and Fas (CD95) expression in CD4+ and in CD8+ T cells in MS patients by using five-color flow cytometry. The percentage of AV+CD4+CD3+ cells and CD95+AV+CD4+CD3+ cells in peripheral blood and cerebrospinal fluid (CSF) were significantly decreased in active MS patients compared with inactive MS patients. A significantly lower proportion of CD95+AV+CD8+CD3+ cells in CSF was observed in active MS patients compared with inactive MS patients, but not in peripheral blood. These results indicate that the resistance of T cells to Fas-mediated apoptosis is involved in exacerbation of MS and/or that Fas-mediated apoptosis of T cells is associated with remission of MS.     

In vitro intercellular adhesion molecule-1 expression on brain endothelial cells in multiple sclerosis

To investigate the origin of intercellular adhesion molecule-1 (ICAM-1) and its expression on brain endothelial cells, we studied the expression in vitro of ICAM-1 on human brain endothelial cells after incubation of T cells from patients with multiple sclerosis (MS) using a histochemical technique and flow cytometry. We determined soluble forms of ICAM-1 (sICAM-1) in the supernatants after mixtures of brain endothelial cells and T cells from patients with MS using an enzyme-liked immunosorbent assay. Flow cytometric analysis showed that a number of ICAM-1-positive cells were significantly increased after incubation of brain endothelial cells with T cells from patients with acute relapsing MS during an exacerbation as compared with those of controls (P<0.01). Patients with acute relapsing MS during an exacerbation and chronic progressive MS exhibited higher levels of ICAM-1 in the supernatants of mixtures with brain endothelial cells and lymphocytes than those of controls (P<0.001 and P<0.01, respectively). These results suggest that lymphocytes from patients with acute relapsing MS during an exacerbation lead to an increased expression of ICAM-1 on the brain endothelial cells and add to evidence involving this adhesion molecule in the pathogenesis of MS.     

Is MS an inflammatory or primary degenerative disease?

Multiple sclerosis (MS) is characterized by multiple areas of inflammation, demyelination and neurodegeneration. Multiple molecular and cellular components mediate neuroinflammation in MS. They involve: adhesion molecules, chemokines, cytokines, matalloproteases and the following cells: CD4+ T cells, CD8+ T cells, B cells, microglia and macrophages. Infiltrating Th1 CD4+ T cells secrete proinflammatory cytokines. They stimulate the release of chemokines, expression of adhesion molecules and can be factors that cause damage to the myelin sheath and axons. Chemokines stimulate integrin activation, mediate leukocyte locomotion on endothelial cells and participate in transendothelial migration. CD8+ cells can directly damage axons. B cells are involved in the production of antibodies which can participate in demyelination. B cells can also function as antigen presenting cells and contribute to T cell activation. Neuroinflammation is not only present in relapsing–remitting MS, but also in the secondary and primary progressive forms of the disease. The association between inflammation consisting of T cells, B cells, plasma cells and macrophages and axonal injury exists in MS patients including the progressive forms of the disease. The above association does not exclude the possibility that neurodegeneration can exist independently from inflammation. Very little inflammation is seen in cortical MS plaques. Anti-inflammatory therapies with different mode of action change the course of MS. Anti-inflammatory and immunomodulatory treatments are beneficial in the early relapsing stage of MS, but these treatments are ineffective in secondary progressive and primary progressive MS. In the stage of progressive MS, inflammation becomes trapped behind a closed or repaired blood–brain barrier. In such a situation current immunomodulatory, immunosuppressive or anti-inflammatory treatments might not reach this inflammatory process to exert a beneficial effect.     

Single-cell analysis of cytokine production shows different immune profiles in multiple sclerosis patients with active or quiescent disease.

Peripheral blood mononuclear cells of multiple sclerosis (MS) patients were stimulated with myelin basic protein (MBP) together with anti-CD28 monoclonal antibody and staphylococcal enterotoxin B to optimize cytokine production by antigen-specific cells. Type 1 (IL-2, IL-12, IFNgamma) and pro-inflammatory (TNFalpha, IL-1beta, IL-6) cytokines were augmented in CD4+, CD8+, and CD14+ cells of acute MS patients and of patients undergoing disease reactivation. These cytokines were reduced in IFNbeta-treated and in stable MS patients; type 2 cytokines (IL-4, IL-10) were increased in these patients. Similar immune profiles are seen in MS patients in whom remission is naturally or pharmacologically (IFNbeta) achieved. Cytokine alterations are particularly evident in CD14+ cells, underlying their critical role in the modulation of the immune response.     

CD45RA+ ICAM-3+ lymphocytes in cerebrospinal fluid and blood as markers of disease activity in patients with multiple sclerosis

OBJECTIVES: Autoreactive T cells targeted against antigens of the myelin sheath are suggested to play an important role in the pathogenesis of multiple sclerosis (MS). Naive (CD45RA+) T cells and intercellular adhesion molecule-3 (ICAM-3) are markers for un-activated lymphocytes. This study was performed to investigate, whether the expression levels of these antigens both on cerebrospinal fluid (CSF) and peripheral blood lymphocytes can be used as activity markers in MS. MATERIALS AND METHODS: Corresponding blood and CSF samples were obtained from 31 patients with relapsing-remitting MS. Of the 31 MS patients 23 were suffering from acute relapses at the time of examination and all of them were treated with high-dose methylprednisolone (MP). Blood was collected again on the 10th day of therapy and after 3 months. The control group consisted of 12 healthy persons. Two-color flow cytometry was performed to evaluate the percentage of both CD45RA+ and ICAM-3+ cells within the lymphocyte population. RESULTS: The percentage of CD45RA+ ICAM-3+ cells in the CSF of MS patients with relapses was significantly increased compared to patients in remission (P<0.05). In blood, a significantly lower percentage of CD45RA+ ICAM-3+ lymphocytes was found in both patient groups compared to healthy controls (Relapse: P<0.05, Remission: P<0.10). Additionally, we found a significant increase (P < 0.01) in the percentage of CD45RA+ ICAM-3+ lymphocytes in blood of MS patients suffering from acute relapse on the 10th day of high-dose MP treatment. CONCLUSION: Our data suggest that the percentage of CD45RA+ ICAM-3+ lymphocytes in CSF can be used as marker of disease activity in MS patients.     

Inhibition of microparticle release triggers endothelial cell apoptosis and detachment

Endothelial cell cultures contain caspase 3-containing microparticles (EMP), which are reported to form during or after cell detachment. We hypothesize that also adherent endothelial cells release EMP, thus protecting these cells from caspase 3 accumulation, detachment and apoptosis. Human umbilical vein endothelial cells (HUVEC) were incubated with and without inhibitors of microparticle release (Y-27632, calpeptin), both in the absence or presence of additional "external stress", i.e. the apoptotic agent staurosporin (200 nM) or the activating cytokine interleukin (IL)-1alpha (5 ng/ml). Control cultures contained mainly viable adherent cells and minor fractions of apoptotic detached cells and microparticles in the absence of inhibitors. In the presence of inhibitors, caspase 3 accumulated in adherent cells and detachment tended to increase. During incubation with either staurosporin or IL-1alpha in the absence of inhibitors of microparticle release, adherent cells remained viable, and detachment and EMP release increased slightly. In the presence of inhibitors, dramatic changes occurred in staurosporin-treated cultures. Caspase 3 accumulated in adherent cells and >90% of the cells detached within 48 hours. In IL-1alpha-treated cultures no accumulation of caspase 3 was observed in adherent cells, although detachment increased. Scanning electron microscopy studies confirmed the presence of EMP on both adherent and detached cells. Prolonged culture of detached cells indicated a rapid EMP formation as well as some EMP formation at longer culture periods. Inhibition of EMP release causes accumulation of caspase 3 and promotes cell detachment, although the extent depends on the kind of "external stress". Thus, the release of caspase 3-containing microparticles may contribute to endothelial cell survival.     

Simvastatin-induced endothelial cell detachment and microparticle release are prenylation dependent

Statins reduce cardiovascular disease risk and affect endothelial function by cholesterol-dependent and independent mechanisms. Recently, circulating (detached) endothelial cells and endothelial microparticles (EMP) have been associated with endothelial functioning in vitro and in vivo. We investigated whether simvastatin affects endothelial detachment and release of EMP. Human umbilical vein endothelial cells (HUVECs) were incubated with clinically relevant concentrations of simvastatin (1.0 and 5.0 microM), with or without mevalonic acid (100 microM) or geranylgeranylpyrophosphate (GGPP; 20 microM) for 24 hours, and analyzed by flowcytometry and Western blot. Simvastatin at 1.0 and 5.0 microM increased cell detachment from 12.5+/-4.1% to 26.0+/-7.6% (p=0.013) and 28.9 +/- 2.2% (p=0.002) as well as EMP release (p=0.098 and p=0.041, respectively). The majority of detached cells was apoptotic, although the fraction of detached cells that showed signs of apoptosis (>70%) was unaffected by simvastatin. Detached cells and EMP contained caspase 3 and caspase 8, but not caspase 9. Restoring either cholesterol biosynthesis and prenylation (mevalonate) or prenylation alone (GGPP) reversed all simvastatin-induced effects on cell detachment and EMP release. Adherent cells showed no signs of simvastatin-induced apoptosis. Simvastatin promotes detachment and EMP release by inhibiting prenylation, presumably via a caspase 8-dependent mechanism. We hypothesize that by facilitating detachment and EMP release, statins improve the overall condition of the remaining vascular endothelium.     

Transforming growth factor beta-1 released from platelets contributes to hypercoagulability in veno-occlusive disease following hematopoetic stem cell transplantation

BACKGROUND: Hepatic veno-occlusive disease (VOD) is one of the most disastrous complications after allogeneic hematopoetic stem cell transplantation (HSCT). Thrombocytopenia with refractoriness to platelet transfusions suggests an increased platelet consumption in these patients. Interactions between platelets and endothelial cells might contribute to the hypercoagulable state at the sinusoidal endothelium as a central mechanism in the pathogenesis of VOD. STUDY DESIGN: The influence of activated platelets on cultured human endothelial cells was investigated in vitro. We focused on the release of plasminogen activator inhibitor-1 (PAI-1) from endothelial cells which has earlier been found to be significantly elevated in plasma of VOD patients. Endothelial cells isolated from human umbilical cords (HUVEC) were incubated with activated platelets. The release of PAI-1 in the presence or absence of specific antibodies was determined by ELISA technique. Tissue factor (TF) expression on endothelial cells was observed by flowcytometric analysis. RESULTS: HUVEC incubated with activated platelets were found to release significantly more PAI-1 compared to untreated cultures. The endothelial PAI-1-secretion after incubation of HUVEC with activated platelets was completely inhibited by an IgG monoclonal antibody against human transforming growth factor beta-1 (TGF beta-1). In contrast, PAI-1 production was not suppressed after inhibition of HUVEC-platelet-interaction by an IgG monoclonal antibody against CD154 (CD40L) expressed on the surface of activated platelets. An increased release of PAI-1 and an increased expression of tissue factor (TF) on the endothelial cell surface were observed after stimulation with TGF beta-1. CONCLUSION: TGF beta-1 released from activated platelets contributes to the hemostatic imbalance at the sinusoidal endothelium in patients with hepatic VOD by increase of endothelial cell PAI-1 production and TF expression. As a potent profibrotic cytokine, TGF beta-1 might further be involved in phlebosclerosis and sinusoidal fibrosis occurring in VOD.     

Reduced adhesion of PBMNCs to endothelium in methylprednisolone-treated MS patients: preliminary results

Methylprednisolone (MP) is a synthetic steroid commonly used in the treatment of multiple sclerosis (MS) relapses. It has a wide spectrum of activities on immune cells: it might also act by preventing mononuclear cell/endothelium adhesion. We studied adhesion phenomena between cultured human umbilical vein endothelial cells (HUVECs) and PBMNCs (CD45⁺, CD14⁺) from 6 MS patients treated in vivo with MP. We also studied fluctuations in CD11a and CD18 levels on lymphocytes and monocytes, as well as changes in serum sICAM-1 and sVCAM-1 concentrations. After MP treatment, PBMNCs adhesion to endothelium decreased at 3 h, while it went back to baseline levels at 24 h. A tendency to increase in both CD11a and CD18 on the surface of lymphocytes was detected, while an increase in serum sVCAM-1 was seen at 3 h.     

Serum from patients with multiple sclerosis downregulates occludin and VE-cadherin expression in cultured endothelial cells

Disruption of the blood brain barrier (BBB) and transendothelial migration of inflammatory cells are crucial steps in the development of demyelinating lesions in multiple sclerosis (MS). Occludin and vascular endothelial-cadherin (VE-cadherin) are two major components of the tight junctions (TJs) in the brain microvasculature that help to create the BBB. In the present study, we investigated the effect of serum from MS patients on the expression of these two junctional markers and on the endothelial integrity. Serum from six MS patients in exacerbation, six in remission, and six normal controls (10% by volume) was incubated with cultured endothelial cells, and the expression of occludin and VE-cadherin was measured by immunoblotting. Serum from MS patients in exacerbation significantly reduced the expression of occludin and VE-cadherin compared with patients in remission and normal controls. This disintegrating effect was more pronounced for occludin than for VE-cadherin. We assume that the elevation in cytokines or other serum-soluble factors in MS patients in exacerbation likely provokes downregulation of occludin and VE-cadherin. This downregulation of TJs proteins may, therefore, contribute to the disruption of the BBB in this condition.