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SummaryRecent studies have shown that peptides identified as surface binding regions of high molecular mass kininogen (HK) and factor XII (FXII) inhibit the Zn2+-dependent binding of FXII to confluent layers of human umbilical vein endothelial cells (HUVEC). This indicates that negatively charged FXII binding surfaces, such as sulfatides and dextran sulfate, may interfere with the binding of FXII to confluent layer of HUVEC. Upon investigating this hypothesis it was unexpectedly found that sulfatides enhanced a specific binding of FXII to a matrix protein expressed during growth of the endothelial cells and that this binding was independent of the presence of Zn2+. The function of sulfatides was partly to minimize nonspecific electrostatic binding and partly to induce and enhance autoactivation of FXII generating αFXIIa. Western blot analysis of the extracts of the matrix incubated with FXII and sulfatides showed that the binding was specific for αFXIIa. The dissociation constant for binding αFXIIa was 12. 8 ± 0. 4 nM (n=4). The binding of αFXIIa to ECM was mapped to the heavy chain as no binding was observed of the light chain containing the catalytic domain. HK, which previously has been shown to completely abolish the Zn2+-dependent binding of FXII to confluent layers of HUVEC, did not inhibit the binding of αFXIIa to the matrix but sulfatides enhanced binding of FXII to ECM. This suggests that HK interferes with the binding of FXII to sulfatides and thereby the autoactivation of FXII. Trypsin treatment of the matrix protein completely abolished the binding, and fibronectin but not laminin was found to bea suitable target. The binding of activated FXII to the ECM suggests that FXIIa may bea modulator of cellular adhesion, migration and vascularization.

SummaryEndothelial progenitor cells (EPC) displaya unique ability to repair vascular injury and promote neovascularization although the underlying molecular mechanisms remain poorly understood. Urokinase-type plasminogen activator (uPA) and its receptor (uPAR) play a critical role in cell migration and angiogenesis by facilitating proteolysis of extracellular matrix.The aim of the present study was to characterize the uPA/uPAR-dependent proteolytic potential of EPC outgrown from human umbilical cord blood and to analyze its contribution to their angiogenic properties in vitro. Cells derived from EPC (EPDC), presenting typical features of late outgrowth endothelial cells, were compared to mature endothelial cells, represented by human umbilical vein endothelial cells (HUVEC). Using quantitative flow cytometry, enzyme-linked immunosorbent assays and zymography, we demonstrated that EPDC displayed higher levels of uPA and uPAR. In conditioned culture media, uPA-dependant proteolytic activity was also found to be significantly increased in EPDC.This activity was paralleled bya higher secretion of pro-metalloproteinase-2 (pro-MMP-2). Inhibition of EPDC-associated uPA by monoclonal antibodies that block either uPA activity or receptor binding, significantly reduced proliferation, migration and capillary like tube formation. Moreover, tumor necrosis factoralpha and vascular endothelial growth factor,known to be locally secreted in ischemic areas, further increased the proteolytic potential of EPDC by up-regulating uPA and uPAR expression respectively.The EPDC response to these factors was found to be more pronounced than that of HUVEC. In conclusion, these findings indicated that EPDC are characterized by high intrinsic uPA/uPAR-dependent proteolytic potential that could contribute to their invasive and angiogenic behaviour.

SummaryAlthough glucocorticoids are widely used in a number of inflammatory disorders associated with endothelial and platelet activation, their effect on the endothelium and platelets in humans remain poorly defined. Hence, we measured changes of a specific endothelial cell marker (von Willebrand factor [vWF]) and of a platelet marker (soluble P-selectin) by infusing therapeutic doses of dexamethasone (0.04 mg/kg and 1.0 mg/kg b.i.d on two days) or placebo into nine healthy men. Venous citrated plasma was obtained before infusion, and at 24 and 48 h. Compared to baseline levels, we found increased levels of vWF at both time points at the higher dose (p=0.011). Plasma levels of sP-selectin rose at 48 h after the high dose (p=0.017). Human umbilical endothelial cells were cultured in the presence or absence of de-xamethasone (0, 0.01, 1 μM), to determine the possible mechanism for the increase in vWF. The vWF-mRNA levels as quantified by RT-PCR increased 2-fold (p

Summary In vitro experiments and animal models indicate that advanced glycation end products (AGEs) may play a crucial role in the vascular dysfunctions observed in patients with diabetes mellitus. These results prompted us to study subrogate markers of inflammation or vascular dysfunction in type II diabetic patients. Monocyte count and activation are dependent upon macrophage colony stimulating factors (M-CSF). Soluble vascular cell adhesion molecule (sVCAM-1) blood levels have been proposed as a marker for endothelium activation. To explore a possible relationship between these factors in diabetic patients, we measured a chemically defined AGE, N(carboxymethyl)lysineprotein (CML-protein) in a group of normal subjects (n = 55) and of diabetic patients (n = 40) using ELISA. Simultaneously, we determined M-CSF and sVCAM-1 blood levels. We found that CML-protein blood levels were significantly higher in patients with diabetes compared to non-diabetic subjects (40.2 ± 4.7 and 7.9 ± 0.7 pmol/mg protein respectively, p < 0.0001). M-CSF was increased while sVCAM-1 blood levels were normal in the group of diabetics. M-CSF blood level was correlated to CML-protein blood level (p < 0.05). In addition CML-protein, M-CSF and sVCAM-1 were increased in patients with microangiopathy. These results suggest that AGE may contribute to vascular dysfunction including microangiopathy.

SummaryThe endothelial progenitor cells (EPCs) are responsible for postnatal vasculogenesis in physiological and pathological neovascularization and have been used for attenuating ischemic diseases. However, EPCs from umbilical cord blood (CB) were not well understood and the homing mechanisms of EPCs remain unclear. To determine the potential application of CB-derived EPCs, we established a culture system to induce the differentiation of CB cells into EPCs. Purified CB CD133+ cells proliferated and, after further vascular endothelial growth factor receptor 2 (VEGFR-2) antibody purification, differentiated into EPCs expressing endothelial markers, such as VE-cadherin, VEGFR-2, CD31, von Willebrand factor (vWF) and WeibelPalade bodies. These cells could also take up acetylated lower density lipoprotein (Ac-LDL) and bind Ulex europaeus agglutinin-1 (UEA-1).When expanded EPCs were transplanted via tail vein into nude mice, they incorporated into capillary networks in ischemic hindlimb, augmented neovascularization, and improved ischemic limb salvage. In addition, in ischemic tissue, there were elevated expressions of VEGF and stromal derived factor 1α???????????(SDF-1α), both of which had chemotactic effect on EPCs. Moreover, P-/E-selectins was found on mouse ischemic endothelium and P-selectin glycoprotein ligand-1 (PSGL-1) on CB-derived EPCs. Neutralizing antibody against PSGL-1 blocked the homing of EPCs to ischemic area by 61%. These results demonstrate that CB CD133+ cell-derived EPCs can be applied for therapeutic neovascularization in ischemic diseases, and reveal important roles of chemoattractants and adhesive molecules in the homing of EPCs.

SummaryEndothelial cell proliferation in response to VEGF plays an important role in physiological and pathological angiogenesis. The role of PDE2 and PDE4 in VEGF-induced proliferation in HUVEC was investigated: 1) VEGF increased cAMP-hydrolytic activity by up-regulating the expression of PDE2 and PDE4 isozymes; 2) VEGF increased progression in cell cycle with an increase in p42/p44 MAP kinase, cyclin A and cyclin D1 expressions and with a decrease in p21waf1/cip1 and p27kip1 expressions; 3) EHNA (20 µM), a selective PDE2 inhibitor, RP73401 (10 µM), a selective PDE4 inhibitor blocked the VEGF-induced increase in p42/p44 MAP kinase expression; 4) RP73401, but not EHNA, blocked the VEGF-induced increase in cyclin A and decrease in p27kip1 expressions; 5) EHNA, contrary to RP73401, enhanced the VEGF-induced increase of cyclin A and decrease of p27kip1. EHNA and RP73401 together blocked the VEGF-induced increase in cyclin D1 and decrease in p21waf1/cip1 expressions; Inhibition of VEGF-upregulated PDE2 and PDE4 reversed the VEGF-induced alterations in cell cycle protein expression, bringing back endothelial cells to a non-proliferating status. Consequently, PDE2 and PDE4 inhibitions were able to inhibit VEGF-induced endothelial cell proliferation by restoring cell cycle key protein expression, and might thus be useful in excessive angiogenesis. Furthermore, the differences between PDE2 and PDE4 effects may suggest compartmentalized effects.

SummaryIn a previous report we have presented evidence that thrombin interacts with αvβ3 integrin in endothelial cells at the molecular and cellular level. This interaction was shown to be of functional significance in vitro and in vivo and contributed to activation of angiogenesis by thrombin. In the present study, we have used a synthetic thrombin peptide, TP508, which represents residues 183 to 200 of human thrombin. This peptide lacks the catalytic site of thrombin but contains the thrombin RGD sequence. Immobilized (surface-coated) TP508 peptide, like thrombin, supported αvβ3 integrin-dependent endothelial cell attachment and haptotactic migration. These effects were specific (a scrambled TP508 peptide was without effect), and dosedependent. The RGD sequence was essential since a modified TP508 peptide, which contained RAD sequence instead of RGD, was inactive. Immobilized TP508 peptide stimulated phosphorylation of mitogen-activated protein kinases and focal adhesion kinase, the signal transduction pathways characteristic for integrin activation. On the other hand, TP508 peptide, when in solution, did not mimic other thrombin-promoted angiogenic effects, such as that of activation gelatinase A, upregulation of expression of vascular endothelial growth factor receptor mRNA or prostacyclin PGI2 release in endothelial cells. On the contrary, soluble TP508 acted as an antagonist for the aforementioned effects of thrombin. TP508 peptide inhibited these thrombin-induced effects through a RGD and α. vβ3-related mechanism. The antagonism with thrombin or thrombin receptor activating peptide was specific and involved at least in part mitogen-activated protein kinases activation. These results point to the importance of RGD sequence of thrombin in mediating effects on endothelial cells and angiogenesis.

SummaryWe examined whether antithrombin (AT) inhibits tumor necrosis factor (TNF)-α-induced endothelial cell activation to elucidate molecular mechanism(s) of the anti-inflammatory activity of AT. AT inhibited the increase in E-selectin expression in cultured human umbilical vein endothelial cells (HUVECs) stimulated with TNF-α. In contrast, chemically modified AT that lacks affinity for heparin did not. AT inhibited the TNF-αinduced interaction of NF-κB p65 with p300, a homologue of cAMP-responsive element binding protein (CREB)-binding protein (CBP). AT increased both intracellular levels of cAMP and binding of phosphorylated-CREB to DNA in HUVECs. Forskolin showed the inhibitory effect similar to that of AT and pretreatment of HUVECs with KT-5720, an inhibitor of protein kinase A, reversed the inhibitory effect of AT. These observations suggested that AT inhibited the TNF-α-induced increase in E-selectin expression in HUVECs by inhibiting the interaction of NF-κB with CBP/p300 through cAMP-dependent protein kinase A-induced CREB activation. This inhibitory activity of AT might depend on its binding to heparin-like substances on the endothelial cell. Such an inhibitory effect of AT on TNF-α-induced endothelial cell activation might at least partly contribute to its anti-inflammatory activity.

SummaryHeparin and low molecular weight heparins (LMWHs) have both antithrombotic and anti-angiogenic activities. The antiangiogenic activity of LMWH may be associated with the release of endothelial tissue factor pathway inhibitor (TFPI), an important endogenous inhibitor of tissue factor/Factor VIIa (TF/fVIIa).To evaluate the effects of LMWH, tinzaparin, and TFPI in a model of angiogenesis-mediated processes, we compared the effects of tinzaparin, and recombinant TFPI in inhibiting either basic fibroblast growth factor-2 (FGF2) or TF/fVIIainduced endothelial cell tube formation in human umbilical vein endothelial cells (HUVEC).Our results show that tinzaparin and recombinant TFPI both blocked endothelial tube formation induced by either FGF2 or TF/fVIIa, in a concentration-dependent manner. Endothelial tube formation was only marginally inhibited by a potent and specific anti-Factor Xa, recombinant tick anticoagulant protein (rTAP). A monoclonal anti-TFPI antibody reversed the inhibitory effects of either tinzaparin or recombinant-TFPI on HUVEC tube formation. Tinzaparin fractions in the range of 8,000 to 12,600 Da were most effective in stimulating the release of TFPI from HUVEC. These results suggest that the inhibitory effect of the LMWH tinzaparin on endothelial tube formation is associated with stimulation of the release of TFPI, but not to anti-Factor Xa activity.

SummaryExcessive leukocyte infiltration causes severe tissue damage in a variety of inflammatory diseases. The initial step in leukocyte extravasation is mediated by selectins and oligosaccharides on their glycoconjugate ligands. Human milk is a rich source of lactose-derived oligosaccharides that are partly absorbed in the intestine and excreted with the urine. As these components contain binding determinants for the selectins we investigated whether human milk oligosaccharides are able to affect leukocyte rolling and adhesion to endothelial cells under dynamic conditions. Therefore, monocytes, lymphocytes, or neutrophils isolated from human peripheral blood were passed over TNF-α-activated HUVEC under shear stress. The influence of different oligosaccharide fractions was determined by video-micros-copy and compared with the effects of various individual oligosaccharides. Within a physiological range (12.5-125 µg/ml) the acidic fraction significantly inhibited leukocyte rolling and adhesion (up to 24.0% and 52.8%, respectively) in a concentrationdependent manner. These effects were even more pronounced than those achieved by soluble sialyl-Lewis x, a physiological binding determinant for selectins. Several active components within the oligosaccharide fraction of human milk were identified, e.g. 3’-sialyl-lactose and 3’-sialyl-3-fucosyl-lactose. These results indicate that specific oligosaccharides in human milk may serve as anti-inflammatory components and might therefore contribute to the lower incidence of inflammatory diseases in human milk-fed infants.

SummaryWe have quantitated the binding of high molecular weight kininogen (HK) to human microvascular endothelial cells of lung and dermal origin as well as to astrocytes and compared the results with those reported for human umbilical vein endothelial cells (HUVEC). We also reassessed parameters of binding to HUVEC employing cells in suspension as well as cells attached to the culture plate and report similar numbers of sites varying from 6.96x105to 7.71x105per cell. The present study shows that HK binds with high specificity and affinity to microvascular endothelial cells (Kd = 1.86 to 4.5 nM) compared to HUVEC (Kd = 10.35nM) but with lower affinity to astrocytes (Kd = 23.73 nM). Human cytokeratin 1, urokinase plasminogen activator receptor and gC1qR were found to be HK binding proteins present at the surface of microvascular endothelial cells and astrocytes analogous to that seen in HUVEC, as assessed by inhibition of binding with antibody to each protein. Lung microvascular endothelial cells had approximately half the number of HK binding sites as HUVEC while dermal micro vascular endothelial cells and astrocytes had only 8-10% of the sites/cell. The affinity of binding to the microvascular endothelial cells was greater than HUVEC, the affinity of binding to astrocytes was considerably less, nevertheless binding to each cell type involves gC1qR, cytokeratin 1 and u-PAR to varying degrees. We also demonstrate, for the first time, that factor XII binds to all of these cell types in a saturable and Zn+2dependent manner. Given that factor XII accelerates the interactions among cell surfaces and proteins of the contact activation cascade to generate bradykinin, binding of factor XII (and the prekallikrein-HK complex) may serve as a mechanism by which these proteins are concentrated locally to facilitate their interactions.

SummaryFibrinogen (fg), present in tumor matrices, has been demonstrated to be determinant in metastatic potential. We have recently shown that fg/ICAM-1 interactions are involved in leukocyte migration across endothelial cell monolayers. Using bladder transitional cell carcinoma as a model, we will show in this study that bladder high grade tumor cell lines express ICAM-1, and that this expression induces an fg-mediated migration. This phenomenon was dependent on ICAM-1/fg interaction as well as RhoA activity. ICAM-1 was concentrated in focal adhesion plaques when tumor cells were allowed to adhere on immobilized fg, suggesting a role in cell migration. The addition of fg induced a 3- to 6-fold enhancement of bladder tumor cell migration through HUVEC monolayers. This process was inhibited by an anti-ICAM-1 antibody blocking fg binding, demonstrating that ICAM-1/fg interaction was involved in the extravasation process. Finally, immunohistological studies revealed that the expression of ICAM-1 was closely associated with an infiltrative histological phenotype.Part of this paper was originally presented as part of the joint meetings of the 16th International Congress of the International Society of Fibrinolysis and Proteolysis (ISFP) and the 17th International Fibrinogen Workshop of the International Fibrinogen Research Society (IFRS) held in Munich, Germany, September, 2002.

SummaryThe regulated expression of adhesion molecules on the surface of endothelial cells is a key process in the pathogenesis of inflammation. The saponin astragaloside IV (AS-IV), a 3-O-β-D-xylopyranosyl-6-O-β-D-glucopyranosylcycloastragenol purified from the Chinese medical herb Astragalus membranaceus(Fisch) Bge.has been shown to have anti-inflammatory effects in vivo.In this study we have investigated the effect of AS-IV on cytokine-and LPS-stimulated expression of adhesion molecules in and leukocyte adhesion to endothelial cells. We have demonstrated that AS-IV significantly reduced the adhesion promoting activity of LPS-stimulated HUVECs for polymorph-nuclear leukocytes (PMNs) and the monocytic cell line THP-1. Furthermore, by using specific cell ELISAs we could show that AS-IV decreased the LPS-induced expression of E-selectin and VCAM-1 on the surface of HUVECs in a dose and time dependent manner, whereas the expression of ICAM-1 was not affected by AS-IV. AS-IV also inhibits TNFβ-induced VCAM-1 expression. The saponin octyl-D-glucopyranoside had no effect on the LPS-induced expression of E-selectin and VCAM-1 excluding an unspecific detergent-like effect of AS-IV. Moreover, AS-IV significantly inhibited LPS- and TNFβ-induced specific mRNA levels for E-selectin and VCAM-1. Finally, we could show that AS-IV completely abolished LPS- and TNFα-induced nuclear translocation of NF-κB and NF-κB DNA binding activity in endothelial cells. We conclude that the ability of AS-IV to inhibit the NF-κB pathway might be one underlying mechanism contributing to its anti-inflammatory potential in vivo.

SummaryAnandamide (AEA), an endogenous cannabinoid, is generated by macrophages during shock conditions, and is thought to be a causative mediator of septic shock. Thus, we hypothesized that AEA plays a crucial role in endothelial cell (EC) injury. Here, we demonstrate that AEA induces apoptosis in a time-and dose-dependent manner in human umbilical vein endothelial cells (HUVECs). AEA triggered phosphorylation of c-Jun NH2-terminal kinase (JNK) and p38 mitogen activated protein kinase. AEA also showed a marked increase of interleukin 1β–converting enzyme (ICE)CED-3 family protease (caspase-3) activity. AEA-induced EC death was inhibited by a selective vanilloid receptor 1 (VR1) antagonist, capsazepine, and was enhanced by a VR1 agonist, capsaicin, indicating that AEA induces apoptosis in ECs via VR1. In conclusion, we propose that AEA may play a crucial role in EC injury under conditions of shock, and that the use of inhibitors of the AEA regulation system may have a therapeutic effect under these conditions.

SummaryThe aminopeptidase inhibitor bestatin has been shown to have anti-angiogenic effects in a number of model systems. These effects are thought to result from inhibition of CD13 activity. Because tumor angiogenesis can evolve in a fibrin-rich stroma matrix we have studied for the first time the effects of bestatin on microvascular endothelial capillary-like tube formation in a fibrin matrix. Bestatin enhanced the formation of capillary-like tubes dose-dependently. Its effects were apparent at 8 µM; the increase was 3.7-fold at 125 µM; while high concentrations (>250 µM), that were shown to have anti-angiogenic effects in other systems, caused extensive matrix degradation. Specific CD13-blocking antibodies WM15 and MY-7, and the aminopeptidase inhibitors amastatin and actinonin also enhanced capillary-like tube formation (maximally 1.5-fold), but these effects did not reach statistical significance. The effect of bestatin was not due to a change in uPAR availability because the relative involvement of the u-PA/u-PAR activity was not altered by bestatin. In view of the present findings we hypothesize that aminopeptidases other than CD13 predominantly contribute to the observed pro-angiogenic effect of bestatin in a fibrin matrix. The identification of this novel effect of bestatin is important in the light of the proposed use of bestatin as anti-angiogenic and/or anti-tumor agent.

SummaryThe exact function of the carbohydrate component of von Willebrand factor (VWF) is unknown. ABO blood group antigens are present as integral structures on the oligosaccharide side chains and it has long been recognised that ABO blood group is a determinant of VWF levels. The mechanism for this is not known. Using a monoclonal antibody against the A-antigen, we investigated the presence of this antigen on VWF from plasma, platelets, human umbilical vein endothelial cells (HUVEC) and saphenous vein endothelial cells. Initial studies on plasma VWF revealed that 23.5% of samples appeared to be negative for the A-antigen. This was shown to correlate with the A2 subtype of the A-antigen (p 2-fold increase in the A-antigen/VWF:Ag ratio for VWF in the plasma. In vitro experiments with serum N-acetlygalactosaminyltransferase failed to demonstrate any addition of A-antigen to platelet or HUVEC VWF. These data are consistent with heterogeneity in the content of A-antigen on VWF from different physiological compartments. Also, they are consistent with either a change in the A-antigen content of VWF after release from the intracellular compartment or a difference in the intracellular addition of A-antigen to VWF by endothelium from different vascular beds.

SummaryThe F11 receptor (F11R) is a cell adhesion molecule (CAM), member of the immunoglobulin superfamily found on the surface of human platelets, and determined to play a role in platelet aggregation, secretion, adhesion and spreading. The same molecule is present also at tight junctions of endothelial cells (EC) where it is known as JAM and acts as a CAM through homophilic interactions. The role of F11R/JAM in the interaction of platelets with endothelial cells was investigated in the current studies. We report here that washed human platelets adhere specifically to a matrix made of immobilized, recombinant sF11R. Furthermore, platelets adhere to cytokine(TNF-α, INF-γ) stimulated human umbilical vein endothelial cells (HUVEC), and approximately 40-60% of the adhesive force is exerted by homophilic interactions between the F11R of platelets and EC. This is evidenced by the inhibition of platelet adhesion to endothelial cells by recombinant soluble form of the F11R, and by two F11R peptides with amino acid sequences of the N-terminal region, and in the 1st Ig fold of the F11R, respectively. This study suggests a role for F11R in the adhesion of platelets to cytokine-inflamed endothelial cells and thus in thrombosis and atherosclerosis induced in non-denuded blood vessels by inflammatory processes. Agents that block the F11R-mediated adhesion of platelets to EC may be of therapeutic value in controlling thrombosis and preventing heart attacks and stroke.

SummaryIn addition to their role in primary hemostasis, platelets serve to support and maintain the vascular endothelium. Platelets contain numerous growth factors including the potent angiogenic inducers VEGF and FGF-2. To characterize the function of these two plateletassociated growth factors, the effects of the addition of purified platelets to cultured endothelial cells were examined. The survival and proliferation of endothelial cells were markedly stimulated (2-3-fold and 5-15-fold respectively) by the addition of gel-filtered platelets. Acetylsalicylic acid-treated or lyophilized fixed-platelets were ineffective in supporting endothelial cell proliferation. In Transwell assays, the stimulatory effect of platelets on endothelial cells was preserved, consistent with an effect mediated by secreted factors. The combined inhibition of VEGF and FGF-2 by neutralizing antibodies, in contrast to inhibition of either alone, abrogated both platelet-induced endothelial cell survival and proliferation. FGF-2 isoforms were detected in platelet lysates, as well as in the releasates of agonist-stimulated platelets. Megakaryocytes generated by ex vivo expansion of hematopoietic progenitor cells with kit ligand and thrombopoietin were analyzed for expression of FGF-2. Punctate cytoplasmic staining but no nuclear staining was observed by immunocytochemistry consistent with possible localization of the growth factor to cytoplasmic granules. The addition of platelets to cultured endothelial cells activated extracellular signal-regulated kinase (ERK) in a dose and time-dependent manner. This effect was abrogated by both anti-FGF-2 and anti-VEGF antibody. Since FGF-2 and VEGF are potent angiogenic factors and known endothelial cell survival factors, their release by platelets provides a plausible mechanism for the platelet support of vascular endothelium.

Tissue factor pathway inhibitor (TFPI) is a protease inhibitor that regulates tissue factor (TF)--initiated coagulation. We report here that cardiomyocytes express TFPI and the expression could be increased by Interleukin-1(IL-1beta). The TFPI expression in cardiomyocytes was confirmed by Northern blotting with rat cardiomyocytes and also by immunostaining with anti-TFPI antibody on human heart specimens from patients either with sarcoidosis, myocarditis or myocardial infarction. The regulation of TFPI expression in cardiomyocytes differs from that in endothelial cells because TFPI expression is not induced in human endothelial cells by IL-1beta.

SummaryWe have investigated the ability of serum from uremic patients to modify the thrombogenic properties of the endothelium. The effects of uremic medium on the morphology of endothelial cells (ECs), and their resistance to flow was analyzed. The influence of uremic media on the reactivity of the extracellular matrix (ECM) generated by ECs towards normal platelets was evaluated in a parallel-plate perfusion chamber. Exposure of ECs to uremic medium resulted in abnormal cell morphology and signs of an accelerated growth. Detachment of ECs exposed to circulating blood was increased when cells had been grown with media supplemented with uremic serum (21% vs. 14% non exposed). Platelet deposition was significantly elevated on ECMs generated in the presence of uremic media (uremicECMs) (p

SummaryFucosylated chondroitin sulfate is a glycosaminoglycan extracted from the sea cucumber Ludwigothurea grisea. This polysaccharide has the same structure as a mammalian chondroitin sulfate but some of the glucuronic acid residues display sulfated fucose branches. Anticoagulant and antithrombotic properties of fucosylated chondroitin sulfate have already been described. In order to further investigate its potential therapeutic use as an antithrombotic agent, we studied its effect on vascular smooth muscle cell (SMC) proliferation and endothelial cell proliferation, migration and Tissue Factor Pathway Inhibitor (TFPI) release. The experiments were performed on SMC from rat thoracic aorta and on human umbilical vein endothelial cell (HUVEC) in culture with or without added fibroblast growth factors (FGF-1 and FGF-2). Our results showed that: (i) fucosylated chondroitin sulfate had a strong inhibitory effect on SMC proliferation (IC50 =10 ± 5 µg/ml) and (ii) no effect on HUVEC proliferation and migration assays, in the absence of exogenous FGF, while heparin had inhibitory effects; (iii) fucosylated chondroitin sulfate (10 µg/ml) enhanced FGF-1 and FGF-2 induced HUVEC proliferation by 45% (145.4 ± 7.2%) and 27% (126.9 ± 4.2%), respectively; (iv) on FGF-induced HUVEC migration, fucosylated chondroitin sulfate (10 µg/ml) had a strong enhancing effect with FGF-1, +122% (222.2 ± 15.8%), three times higher than that of heparin, and a lower enhancing effect with FGF-2, +43% (142.7 ± 4.6%), whereas heparin had no effect; (v) fucosylated chondroitin sulfate stimulated TFPI release, mainly on the free form, +98% (198.2 ± 25.%). In addition, the structural features of the polysaccharide associated with its biological activity were resolved using chemically modified fucosylated chondroitin sulfates. Sulfated fucose branches groups are essential to the potentiating effect of the polysaccharide on HUVEC proliferation and migration. Surprisingly, removal of fucose branches from the fucosylated chondroitin sulfate did not abolish TFPI release. Finally, partial reduction of the glucuronic acid carboxyl groups limited the potentiating effect on HUVEC proliferation and migration but did not affect TFPI release. In conclusion, this fucosylated chondroitin sulfate from invertebrate origin reveals useful properties for an antithrombotic agent: inhibition of SMC proliferation, enhancement of endothelium wound repair and TFPI release. These properties on vascular cells, associated with a low bleeding tendency and an antithrombotic activity, strongly suggest its potential use as a new therapeutic agent in arterial thrombosis and restenosis, with a more favorable effect than heparin.

SummaryThrombin interacts with its receptor and thrombomodulin on endothelial cells. We evaluated the respective roles of these two proteins on human umbilical vein endothelial cell (HUVEC) growth by comparing thrombin, S195A (a mutant thrombin in which the serine of the charge stabilizing system had been replaced by alanine), and the receptor activating peptide (TRAP). Thrombin and TRAP induced DNA synthesis (half maximal cell proliferation with 5 nM and 25 μM, respectively), whereas S195A thrombin was inactive, inferring that growth is mediated through the thrombin receptor. Surprisingly, cells stimulated by TRAP exhibited a maximal proliferation twice greater than that obtained with thrombin. Combination of thrombin and TRAP resulted in a mitogenic response higher than by thrombin alone, but lower than by TRAP alone. The role of thrombomodulin was evaluated by adding an anti-thrombomodulin antibody, which prevents formation of the thrombin-thrombomodulin complex. Antibody did not interfere with cell proliferation induced by TRAP, but enhanced that induced by thrombin. We conclude that formation of the thrombin-thrombomodulin complex restrains HUVEC proliferation mediated through the thrombin receptor.

SummaryWe investigated the effect of heat shock on the fibrinolytic potential of human umbilical vein endothelial cells (HUVECs) in culture. When cultured at 43° C, the mRNA for heat shock protein 70 (HSP70) was dramatically induced within 120 min with a maximal induction of more than 90-fold compared with that in HUVECs cultured at 37° C. The level of urokinase-type plasminogen activator (u-PA) receptor (u-PAR) mRNA increased up to 2.2-fold in response to heat shock, which was associated with the increased u-PA binding and cell-surface u-PA activity determined by adding exogenous u-PA to acid-treated HUVECs. The increased u-PAR mRNA returned to normal level when HUVECs were further incubated at 37° C for 180 min, and this decline was not affected in the presence of actinomycin D. Though the secreted antigens for tissue-type plasminogen activator (t-PA) and type 1 plasminogen activator inhibitor (PAI-1) in the conditioned medium (CM) of HUVECs were simultaneously increased at 43° C during this period, the increase in the levels of t-PA (about 26.6-fold at 120 min) was greater than that of PAI-1 (1.8-fold at 120 min). The fibrinolytic activity of CM obtained from HUVECs at 43° C was significantly enhanced up to 3-fold, indicating that heat shock induced hyperfibrinolytic states in HUVECs. The secretion of u-PA into CM was also enhanced by heat shock. These results suggested that human endothelial cells respond to hyperthermia by inducing HSP70 followed by hyperfibrinolytic states with the enhanced expression of u-PAR as well as that of t-PA and u-PA.

SummaryFactor XII has long been implicated in the intrinsic pathway of fibrinolysis, but the mechanism by which it triggers plasminogen activation and targets fibrinolysis has not been established. In the present study, the assembly and function of activated Factor XII (F.XIIa), prourokinase (pro-u-PA), high molecular weight kininogen (H-kininogen), and prekallikrein on human umbilical vein endothelial cells (HUVEC) was investigated. 125I-prekallikrein was shown to bind to HUVEC via receptor-bound H-kininogen in the presence of 50 μM ZnCl2. After the addition of F.XIIa, 78% of the 125I-prekallikrein initially bound to HUVEC was converted to 125I-kallikrein. However, only 6% of the HUVEC-bound 125I-pro-u-PA was thereby activated. This discrepancy was shown to be related to rapid dissociation (>50% within 15 min) of prekallikrein/kallikrein, but not pro-u-PA, from HUVEC. Increasing the level of cell-bound kallikrein increased the portion of cell-bound pro-u-PA activated, indicating that their co-localization was important for this pathway. Finally, F.XIIa was shown to trigger plasminogen activation on HUVEC via this pathway. This assembly of reactants on the endothelium suggests a mechanism whereby local fibrinolysis may be triggered by blood coagulation.

SummaryDuring pregnancy, important modifications of hemostasis occur resulting in mothers in hypercoagulability and the role of placental cells such as trophoblast cells has been hypothesized. In this study, we first showed that syncytiotrophoblast plasma membranes, isolated from normal human placenta, expressed a strong tissue factor (TF) activity. We then studied TF activity of two continuous trophoblast cell lines (JEG-3 and BeWo) in comparison to human umbilical vein endothelial cells (HUVEC) and transformed human endothelial cells (ECV-304). TF assays were performed on intact detached confluent cells. Unstimulated JEG-3 and BeWo cells exhibited a very high TF activity which slightly increased after 2 to 4 h TNF-α stimulation. In contrast, HUVEC and ECV-304 had a lower basal TF activity which was mainly inducible by TNF-a, with a maximum effect after 4 to 6 h stimulation. For both cell types, TF activity was decreased to basal value after 16-hour TNF-α stimulation. These results support that trophoblast cells are able to express TF but the involvement of this property in the hemostatic physiological changes observed during pregnancy, remains to be demonstrated.

SummaryThe ability of vanadate, an inhibitor of protein-tyrosine phosphatases, to affect the production of prostacyclin (PGI2) and endothelin-1 (ET-1) and protein-tyrosine phosphorylation in human umbilical vein endothelial cells (HUVEC) was studied. The addition of vanadate to monolayers of cultured HUVEC caused a sustained release of PGI2 from HUVEC in a time- and dose-dependent manner. When aspirin-treated HUVEC, which have lost the ability to increase PGI2 production in response to arachidonate, were incubated with vanadate, the cells recovered their ability to increase PGI2 production in response to arachidonate. This recovery of inducible PGI2 production in aspirin-treated HUVEC was completely inhibited either by cycloheximide, a protein synthesis inhibitor, or by actinomycin D, an RNA synthesis inhibitor. In contrast, the same concentration of vanadate suppressed the basal release of ET-1 from HUVEC. Vanadate also caused an increase in protein-tyrosine phosphorylation in HUVEC. These data indicate that vanadate induces opposite effects on PGI2 and ET-1 production with a concomitant increase in protein-tyrosine phosphorylation in HUVEC.

SummaryCultured human umbilical vein endothelial cells were incubated with defibrotide at concentrations of 0, 5, 50 and 500 jxg/ml for 4 and 24 h respectively. Thrombomodulin activity and molecules on the surface of the cells were determined by chromogenic assay and radioimmunoassay, thrombomodulin antigen in endothelial cells and in conditioned medium of the cells was measured by immunoradioassay. Thrombomodulin mRNA within the cells was analysed by slot blot. After 24 h of incubation, the activity and molecules of thrombomodulin on the surface of endothelial cells, as well as the antigen and mRNA of thrombomodulin in the cells were significantly increased in a dose dependent manner. However, the level of thrombomodulin antigen in conditioned medium was about equal to that of the control. Our data indicate that defibrotide stimulates expression of thrombomodulin in human endothelial cells. These beneficial effects may play a role in antithrombotic activity of defibrotide.

SummaryWe quantified thrombin-induced endothelial cells shape change and investigated the role of Ca2+ in such shape change. We used the fluorescent Ca2+ indicator, fura2, to measure both shape change as cell size and intracellular free Ca2+ ([Ca2+]i), in cultured human umbilical-vein endothelial cells (HUVEC). Thrombin induced concentration-dependent decreases in cell size (percentage of cell size at 6 min after stimulation with 0.01 U/ml, 0.1 U/ml, or 1 U/ml thrombin) was 90.1 ± 1.5%, 78.1 ± 2.4%, and 40.9 ± 2.4%, respectively. Thrombin also increased [Ca2+]i in a concentration-dependent manner. Both depletion of extracellular Ca2+, and also the addition of W5, a calmodulin antagonist, inhibited thrombin-induced size reduction. These results indicate an association between shape change and [Ca2+]i mobilization in human endothelial cells stimulated by thrombin.

Integrins alphavbeta3 and alphavbeta5 have been long considered as proangiogenic receptors, yet genetic ablation studies demonstrated enhanced angiogenesis in mice lacking alphavbeta3 and alphavbeta5 integrins, which was attributed to increased expression of vascular endothelial growth factor receptor-2 (VEGFR-2). In this study, we determined the effect of alphavbeta3 and alphavbeta5 suppression in endothelial cells (EC) on vascular endothelial growth factor (VEGF) and VEGFR-2 expression. alphav was suppressed by shRNA in HUVEC (venous endothelial cells) and HMVEC (microvascular endothelial cells). VEGFR-2 was significantly upregulated by alphav suppression in HUVEC and downregulated in HMVEC at both mRNA and protein levels, as assessed by real-time PCR and flow cytometry, respectively. HMVEC displayed completely abolished Sp1 binding to the VEGFR-2 promoter, and HUVEC exhibited enhanced binding to the -170 E-Box element in the VEGFR-2 promoter, assessed by electrophoretic mobility shift assay. Realtime PCR also revealed opposite effects on the expression of 5 additional important genes involved in angiogenesis in the two cell types. VEGF mRNA expression was enhanced in HUVEC and reduced in HMVEC; however, these alterations were not statistically significant. VEGF-induced proliferation was upregulated in HUVEC and reduced in HMVEC following alphav suppression. No tube formation on Matrigel was observed in alphav suppressed cells, regardless of their origin. These results indicate that suppression of alphav integrins can either augment or inhibit VEGFR-2 levels and VEGF-induced proliferation in EC from different vascular beds. Hence, therapeutic antiangiogenic intervention by siRNA- mediated suppression of alphav integrins should take into account variable and potentially hazardous responses in different vascular beds.

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.

Monocytes and platelets are both crucially involved in atherogenesis. Importantly, activated platelets bound to circulating monocytes increase adhesion of the monocytes and thus mediate colocalization of both cell types at the vessel wall. We examined the fate of the platelets upon migration of these potentially pro-atherogenic platelet-monocyte complexes (PMC) across activated endothelium. Platelet-monocyte complex migration was studied both quantitatively by means of Transwell filters coated with endothelial cells, as well as qualitatively with different imaging techniques, and in the absence or presence of flow. Upon PMC transendothelial migration, platelets relocate with monocytic P-selectin glycoprotein ligand-1 (PSGL-1) to the rear of the monocyte, detach, and remain at the endothelial surface. Platelet dissociation appeared not to be due to reduced PSGL-1 expression or reduced platelet-binding capacity of the migrated monocytes. In addition, different endothelial matrix proteins with different platelet-binding capacities coated on the Transwell filter, instead of endothelial cells, did not affect PMC dissociation. In contrast, lowering the mechanical stress that PMC experience during transmigration prevented dissociation of platelets. In conclusion, PMC dissociate during transendothelial migration as a result of monocytic PSGL-1 redistribution and mechanical stress. PMC-mediated deposition of activated platelets at sites of vascular inflammation is likely relevant for cardiovascular disease progression or vascular regeneration.

Tissue factor (TF) plays a pivotal role in thrombus formation and atherogenesis in acute coronary syndrome. Tissue factor pathway inhibitor (TFPI) is a specific physiological inhibitor of TF/FVIIa complex that regulates TF-induced coagulation. Adiponectin (Adp) is an adipocyte-specific adipocytokine with anti-atherogenic and anti-diabetic properties. Adp inhibits inflammatory cytokine and adhesion molecules expression, and it can prevent endothelial dysfunction. In this study, we investigated the effects of Adp on tumor necrosis factor-alpha (TNF-alpha)-induced expression of TF and TFPI in human umbilical vein endothelial cells (HUVECs), and the signaling transduction pathways involved. It was found that Adp significantly inhibited both TF protein expression and activity in TNF-alpha-stimulated HUVECs. In the meanwhile, it increased TFPI protein expression and activity for about two folds. Adp also inhibited TF mRNA expression induced by TNF-alpha, but had no effect on TFPI mRNA expression. The inhibitory effect of Adp on TNF-alpha-induced TF expression was prevented by pretreatment with Rp-cAMPs, a PKA inhibitor. Adp increased intracellular cAMP content and PKA activity levels in a dose-dependent manner. Phosphorylation of IkappaB-alpha was decreased by Adp, but phosphorylation of p44/42 MAPK, SAPK/JNK, and p38 MAPK were not affected. These results suggested that Adp inhibits TF expression through inhibition of a PKA dependent nuclear factor-kappaB (NF-kappaB) signaling pathway. It was also found that adiponectin promoted Akt and AMP-activated protein kinase phosphorylation. The inhibitory effect of Adp on TNF-alpha-induced TF synthesis was abrogated in part by pretreatment with the PI3kinase inhibitor LY294002, suggesting that Akt activation might inhibit TF expression induced by TNF-alpha. The inhibitory effect of Adp is almost completely abrogated by inhibition of both the cAMP/PKA pathway and PI3K/Akt pathway. In conclusion, our data indicated that inhibition of NF-kappaB through stabilization of IkappaB-alpha and activation of Akt phosphorylation may mediate the inhibitory effect of Adp on TF expression; but the enhancement effect of Adp on the TFPI production might occur via translational rather than transcriptional regulation.

SummaryDespite the success of the anti-coagulant protease protein C (PC) in treating septic shock in humans, the signaling pathways used are still unclear. To explore the effects of treatment with PC zymogen and its activated form aPC in a setting of sepsis, we employed a piglet model of endotoxic shock. In the aPC group, we observed a 65%-90% reduction in plasmaTNF-alpha levels and a concomitant clinical improvement. Unexpectedly, administration of aPC also resulted in stabilization of the plasma pH above 7.2. Moreover, phosphorylated p38 mitogen-activated protein kinase (p38MAPK) was virtually absent in the livers of those piglets receiving aPC. In cultured human umbilical vein endothelial cells, we observed that nanomolar concentrations of PC and aPC inhibited the phosphorylation of p38MAPK. Furthermore, we showed that the regulation of the pro-apoptotic cell cycle regulator p53 by PC and aPC is dependent on the reduction of p38MAPK activation. The transduction of these effects involves all three receptors associated with protein C signaling, namely endothelial protein C receptor, protease-activated receptor 1, and sphingosine 1-phosphate receptor 1. Ultimately, this study elucidates novel signaling pathways regulated by protein C and emphasises the pivotal importance of its multiple modes of action beyond anticoagulation. APC’s clinical success may, in part, be due to p38MAPK inhibition.

SummaryEndothelial 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)-1α (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-1α 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-1α-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.

The acute physiologic release of tissue-type plasminogen activator (t-PA) from the endothelium is critical for vascular homeostasis. This process is prostacyclin- and nitric oxide (NO)-independent in humans. It has been suggested that calcium signaling and endothelial-derived hyperpolarizing factors (EDHF) may play a role in t-PA release. G-protein-coupled receptor-dependent calcium signaling is typically Galphaq-dependent. EDHFs have been functionally defined and in various tissues are believed to be various regioisomers of the epoxyeicosatrienoic acids (EETs). We tested the hypothesis in vitro that thrombin-stimulated t-PA release from human microvascular endothelial cells (HMECs) is both Galphaq- and EDHF-dependent. Conditioned media was harvested following thrombin stimulation, and t-PA antigen was measured by ELISA. Thrombin-induced t-PA release was limited by a membrane-permeable Galphaq inhibitory peptide, the PLC-beta antagonist U73122, and the IP3 receptor antagonist 2-aminoethoxyphenylborane, while the Galphaq agonist Pasteurella toxin modestly induced t-PA release. The cytochrome P450 (CYP450) inhibitor, miconazole, and the arachidonic acid epoxygenase inhibitor MS-PPOH inhibited thrombin-stimulated t-PA release, while 5,6-EET-methyl ester stimulated t-PA release. The 5,6- and 14,15-EET antagonist, 14,15-epoxyeicosa-5(Z)-enoic acid, inhibited t-PA release at the 100 microM concentration. However, thrombin-stimulated t-PA release was unaffected by the prostacyclin and NO inhibitors ASA and L-NAME, as well as the potassium channel inhibitors TEA, apamin and charybdotoxin. These studies suggest that thrombin-stimulated t-PA release is Galphaq-, PLC-beta-, IP3-, and 5,6-EET-dependent while being prostacyclin-, NO- and K+ channel-independent in HMECs.

Expression of isolated beta integrin cytoplasmic domains in cultured endothelial cells was reported to induce cell detachment and death. To test whether cell death was the cause or the consequence of cell detachment, we expressed isolated integrin beta1 cytoplasmic and transmembrane domains (CH1) in cultured human umbilical vein endothelial cells (HUVEC), and monitored detachment, viability, caspase activation and signaling. CH1 expression induced dose-dependent cell detachment. At 24 h over 90% of CH1-expressing HUVEC were detached but largely viable (85%). No evidence of pro-caspase-8,-3, and PARP cleavage or suppression of phosphorylation of ERK, PKB and Ikappa-B was observed. The caspase inhibitor z-VAD did not prevent cell detachment. At 48 h, however, CH1-expressing cells were over 50% dead. As a comparison trypsin-mediated detachment resulted in a time-dependent cell death, paralleled by caspase-3 activation and suppression of ERK, PKB and Ikappa-B phosphoyrylation at 24 h or later after detachment. HUVEC stimulation with agents that strengthen integrin-mediated adhesion (i.e. PMA, the Src inhibitor PP2 and COMP-Ang1) did not prevent CH1-induced detachment. Expression of CH1 in rat carotid artery endothelial cells in vivo caused endothelial cell detachment and increased nuclear DNA fragmentation among detached cells. A construct lacking the integrin cytoplasmic domain (CH2) had no effect on adhesion and cell viability in vitro and in vivo. These results demonstrate that isolated beta1 cytoplasmic domain expression induces caspase-independent detachment of viable endothelial cells and that death is secondary to detachment (i.e. anoikis). They also reveal an essential role for integrins in the adhesion and survival of quiescent endothelial cells in vivo.

SummarySevere sepsis in children or adults may cause a life-threatening coagulopathy, with widespread consumption of activated protein C (APC); recombinant human APC (rhAPC) is a promising candidate anticoagulant treatment. We investigated the effects of rhAPC and other anticoagulants on coagulation triggered by adding small quantities of lipidated tissue factor to human umbilical-cord plasma in vitro. rhAPC, unfractionated heparin (UH),and melagatran (a direct thrombin inhibitor) were studied individually, and in combinations of rhAPC with either UH or melagatran. rhAPC alone dose-dependently prolonged the activated partial-thromboplastin time (aPTT) but not the prothrombin time (PT), and dose-dependently suppressed two indices of thrombin generation, namely prothrombin fragment F 1.2 (F 1.2) generation and thrombin–antithrombin (TAT) complex formation. UH alone dose-dependently prolonged the aPTT but not the PT, while melagatran alone dose-dependently prolonged both the aPTT and the PT. Adding either UH or melagatran dose-dependently augmented the capacity of rhAPC to suppress F 1.2 generation (with addition of UH showing a greater effect) and TAT formation (with addition of melagatran showing a greater effect). Both the capacity of UH to prolong the aPTT and the capacity of melagatran to prolong the aPTT and the PT were augmented by adding rhAPC. In our in-vitro study, adding either UH or melagatran augmented the capacity of rhAPC to suppress thrombin generation in human umbilical-cord plasma, with the anticoagulant effect of melagatran being more predictable than that of UH. Hence, combining rhAPC with melagatran might be a valuable therapeutic option in patients with severe sepsis.

Summary Streptococcus pneumoniae is the major pathogen of communipnetyacquired umonia and a common cause of otitis, meningitis and sepsis. During pneumococci infection accompanied with bacterial invasion and hematogenous spreading, the endothelium is directly targeted by pneumococci and their virulence factors. Therefore, we tested the hypothesis that pneumococci induced endothelial apoptosis. Unencapsulated R6x pneumococci strongly induced apoptosis of human endothelial cells both from lung microvasculature and umbilical vein, whereas an encapsulated strain D39 mainly led to necrotic cell death. Deletion of the gene coding for pneumolysin reduced pneumococci-induced apoptosis in HUVEC. Furthermore, N-acetyl-L-cysteine, an antioxidant thiol, significantly reduced apoptosis caused by R6x, and LDH release induced by D39, pointing to a role for reactive oxygen species in the pathogenesis. Apoptotic cells showed increased cleavage and activity of caspases 6 and 9 but only late activation of caspase 3. Programmed cell death could be strongly reduced by pan-caspase inhibitor zVAD. Reduced levels of Bcl2 and cytosolic increase of apoptosis-inducing factor in pneumococci-infected cells implicated involvement of mitochondrial death pathways. Caspase activation and apoptosis were abolished by cAMP elevation. Moreover, p38 mitogen-activated protein kinase and c-Jun NH2-terminal kinase were activated in pneumococci-infected cells and inhibitors of both kinases strongly reduced pneumococci-induced caspase activation and apoptosis. Hence,kinase- and caspase-dependence of pneumococci-induced endothelial apoptosis may bear relevance to novel therapeutic approaches to pneumococci-related disease.

SummaryExtracellular nucleotides bind to type-2 purinergic/pyrimidinergic (P2) receptors that mediate various responses, such as cell activation, proliferation and apoptosis, implicated in inflammatory processes. The role of P2 receptors and their associated signal transduction pathways in endothelial cell responses has not been fully investigated. Here, it is shown that stimulation of human umbilical vein endothelial cells (HUVEC) with extracellular ATP or UTP increased intracellular free calcium ion concentrations ([Ca2+]i), induced phosphorylation of focal adhesion kinase (FAK), p130cas and paxillin, and caused cytoskeletal rearrangements with consequent cell migration. Furthermore, UTP increased migration of HUVEC in a phosphatidylinositol 3-kinase (PI3-K)-dependent manner. BAPTA or thapsigargin inhibited the extracellular nucleotide-induced increase in [Ca2+]i, a response crucial for both FAK phosphorylation and cell migration. Furthermore, long-term exposure of HUVEC to ATP and UTP, agonists of the G protein-coupled P2Y2 and P2Y4 receptor subtypes, caused upregulation of αv integrin expression, a cell adhesion molecule known to directly interact with P2Y2 receptors. Our results suggest that extracellular nucleotides modulate signaling pathways in HUVEC influencing cell functions, such as cytoskeletal changes, cellular adhesion and motility, typically associated with integrin-activation and the action of growth factors. We propose that P2Y2 and possibly P2Y4 receptors mediate those responses that are important in vascular inflammation, atherosclerosis and angiogenesis.

SummaryPm-VEGF, a novel member ofVEGF family from the venom gland of Taiwan habu (Protobothrops mucrosquamatu), is a disulfidelinked homodimer with 119 amino acid residues. Recombinant fusion Pm-VEGF was expressed in Escherichia coli, purified and refolded. Surface plasmon resonance was used to determine its binding kinetics toVEGF-receptors (VEGFR). Relative to human VEGF165, the binding affinity of Pm-VEGF to the VEGFR-1 was 1.7-fold higher while affinity to the VEGFR-2 was 17-fold lower. But it did not bind theVEGFR-3 or neuropilin-1. Pm-VEGF promoted the proliferation and tissue factor production of endothelial cells, the neovascularization in the chicken chorioallantoic membrane, and increased vascular permeability. It also stimulated tissue-factor production and human monocyte chemotaxis, in accord with its specificity for VEGFR-1. Structural comparison among VEGF-proteins from various viper venoms revealed that the two subfamilies of vipers (Crotalinae and Viperinae) have evolved with distinct receptor-specificities for VEGFR-1 and VEGFR-2, respectively. Discussion on structureactivity relationships of the VEGFs further provided insight into residues important for the receptor-binding and specificities.

SummaryActivation of plasma prekallikein and generation of bradykinin are responsible for the angioedema attacks observed with C1inhibitor deficiency. Heterozygous individuals with

SummaryCa2+-activated K+ channels with large conductance (BKCa) have been shown to play an important role in the regulation of vascular tone. We examined the role of the p42/p44 MAP-kinase (p42/p44MAPK) on nitric oxide (NO) production in human endothelial cells induced by the BKCa-opener NS1619. Using DiBAC-fluorescence imaging a concentration-dependent (2.5-12.5 µM) hyperpolarization induced by NS1619 was observed. A significant increase of intracellular Ca2+-concentration by NS1619 was seen using Fura-2-fluorescence-imaging, which was blocked by 2-APB, or reduction of extracellular Ca2+ (n=30; p

The objective of this study was to characterize the role of fibrinogen in stimulating expression of inflammatory chemokines in endothelial cells through NF-kappaB activation. Human umbilical vein endothelial cells (HUVEC) were exposed to fibrinogen up to 3,000 microg/ml, and NF-kappaB activation was assessed using electrophoretic mobility shift assay (EMSA). Fibrinogen exposure resulted in a concentration dependent increase in NF-kappaB activation that reached a maximum at 1,000 microg/ml after 4 hours and was sustained up to 24 hours. The effect was inhibited by antibodies to alpha(v)beta(3) and alpha(5)beta(1) and by the GRGDS peptide, indicating integrin involvement. Preincubation with Mn(2+) lowered the fibrinogen concentration-dependence, consistent with integrin activation. Supershift assays demonstrated involvement of the p50, p65 and c-Rel components of NF-kappaB. Fibrinogen exposure also resulted in up-regulation of expression of monocyte chemoattractant protein-1 (MCP-1) and of interleukin-8 as shown by RNase protection assays and by real-time RT-PCR. Increased secretion of MCP-1 was confirmed by ELISA. Parthenolide, an IkappaB kinase inhibitor, prevented up-regulation of MCP-1 by fibrinogen, linking this response to NF-kappaB activation. From our findings, we conclude that fibrinogen regulates NF-kappaB activation and expression of inflammatory chemokines in endothelial cells and may be involved in mediating inflammatory processes.

SummaryThe placenta is a highly vascularized organ with fetal and maternal blood supply. Syncytiotrophoblasts (STB), which line the placenta villous are possibly involved in local hemostatic mechanisms.The aim of this study was to evaluate the levels of tissue factor (TF) and its inhibitors, tissue factor pathway inhibitor (TFPI,TFPI-2), in STB model within hemostatic and inflammatory environments. Human primary STB cell cultures were characterized by vascular and hormonal markers. TF and TFPI mRNA expression, protein levels and activity were determined and compared to human umbilical vein endothelial cells (HUVEC). High levels of TF were demonstrated in STB cells compared to low levels in HUVEC. In contrast, STB expressed lower TFPI levels than HUVEC. LPS and TNFα increased the high constitutive TF in STB, whereas LPS and IL-1α further reduced TFPI levels. The procoagulant tendency of STB identified by us may reflect the physiological need for immediate inhibition of hemorrhage in the placental inter-villous spaces in basal and inflammatory conditions.This hemostatic balance may be critical for normal placental function and pregnancy outcome.

SummaryThe anti-angiogenic properties of the histidine-proline–rich (H/P) domain of HPRG have recently been described (Juarez JC, et al. Cancer Research 2002; 62: 5344-50). However, the binding site that mediates these properties is unknown. HPRG is evolutionarily, functionally and structurally related to cleaved high molecular weight kininogen (HKa), an anti-angiogenic polypeptide that stimulates apoptosis of proliferating endothelial cells through binding to cell-surface tropomyosin (Zhang J-C, et al. Proc Natl Acad Sci USA 2002; 99: 12224-9). In this study, we demonstrate that HPRG binds with high affinity to FGF-2–stimulated human umbilical vein endothelial cells (HUVEC) and immobilized tropomyosin in a Zn2+ or pH-dependent manner, and that this interaction is mediated by the H/P domain of HPRG. At least two binding sites for HPRG, tropomyosin and heparan sulfate proteoglycans (HSPs), were identified on the surface of FGF-2–activated endothelial cells. Translocation of tropomyosin to the surface of HUVEC occurred in response to FGF-2, and the anti-angiogenic activity of HPRG in a Matrigel plug model was partially inhibited by soluble tropomyosin. These results suggest that HPRG binds to endothelial cell surface tropomyosin which at least partially mediates the antiangiogenic effects of HPRG.

SummaryAfter high-voltage electric injury, patients often show tissue necrosis and thrombosis of blood vessels even remote from entry and exit site of electrical current. In this study, plasma levels of TAT, F1+2, PAI-1, and t-PA were determined in vivo in three patients with high-voltage injury for 96 hours after trauma. In order to analyse a possible effect on haemostasis related to endothelial cell damage, protein S, TF, ET-1, PGI2, NO, t-PA, and PAI-1 were determined for 72 hours in vitro in cell culture supernatant of HUVECs that had been exposed to 1, 10, 30, and 50 electric field periods of 50 Hz with field strength of 60 V/cm and duration of 20 ms. Furthermore, expression of thrombomodulin was immunohistochemically analysed. Clotting activation could be observed in our patients by increased levels of F1+2 and TAT between 12 and 72 hours after injury, whereas fibrinolysis was disturbed due to high PAI-1. One patient presented thrombosis of vessels by day 3. In vitro, PAI-1 increased significantly (p

Drotrecogin alfa (activated) prolongs half-life time of messenger RNA encoding for Interleukin-8 and Monocyte chemoattractant protein-1

Intravenous gamma-immunoglobulin (i.v.Ig) is commonly used in the treatment of autoimmune and inflammatory vascular disorders to prevent thrombotic complications. The mechanism of action of i.v.Ig is, however, not yet elucidated. In view of this, we investigated the ability of i.v.Ig to modulate i) Ca(2+) signals of fura-2 loaded endothelial cells, and ii) the associated release of nitric oxide (NO) and von Willebrand factor (vWf). NO was measured either indirectly by radioimmunoassay of cGMP in unstimulated cells or directly by electrochemistry at the surface of stimulated endothelial cells from human umbilical cord veins (HUVEC). Short-term treatment of unstimulated HUVEC with intact i.v.Ig decreased the basal cytosolic Ca(2+) concentration by 20% while it activated the NO/cGMP synthesis. Following i.v.Ig treatment of HUVEC, the Ca(2+) liberation from internal stores and the vWf secretion induced by ATP, thrombin or histamine were significantly reduced by 38 and 60%, respectively. The effects on Ca(2+) signals were observed with intact i.v.Ig as well as with the F(ab')2 or the Fc fragments indicating that both portions are involved in the mechanism of action. The i.v.Ig treatment of HUVECs had no effect on the NO release induced by thrombin or histamine. By contrast, the i.v.Ig treatment increased the ATP-activated NO release by amplifying the Ser1177-eNOS phosphorylation. The i.v.Ig also activated the NO-dependent cGMP release in resting and collagen-stimulated platelets. Since NO is a potent inhibitor of platelet activation and vWF is a platelet adhesion cofactor, the beneficial effects of therapeutic i.v.Ig may lie in the inhibition of platelet adhesion to damaged endothelium.

SummaryMigration and proliferation of endothelial cells in response to VEGF play an important role in angiogenesis associated to pathologies such as atherosclerosis, diabetes and tumor development. Elevation of cAMP in endothelial cells has been shown to inhibit growth factor-induced proliferation. Our hypothesis was that inactivation of cAMP-specific phosphodiesterases (PDEs) would inhibit angiogenesis. The purpose of this study was to evaluate the effect of PDE inhibitors on in vitro and in vivo angiogenesis, using human umbilical vein endothelial cell (HUVEC) and chick chorioallantoic membrane (CAM) models respectively. Here, we report that: 1) PDE2, PDE3, PDE4 and PDE5 are expressed in HUVEC; 2) EHNA (20 µM), PDE2 selective inhibitor, and RP73401 (10 µM), PDE4 selective inhibitor, are able to increase the intracellular cAMP level in HUVEC; 3) EHNA and RP73401 are able to inhibit proliferation, cell cycle progression and migration of HUVEC stimulated by VEGF; 4) these in vitro effects can be mimic by treating HUVEC with the cAMP analogue, 8-Br-cAMP (600 µM); 5) only the association of EHNA and RP73401 inhibits in vivo angiogenesis, indicating that both migration and proliferation must be inhibited. These data strongly suggest that PDE2 and PDE4 represent new potential therapeutic targets in pathological angiogenesis.

The activated protein C (APC) pathway has been suggested to be a common link between coagulation and inflammation. APC may function to restore hemostasis via modulation of cytokine expression. We investigated the effect of APC on the endothelial expression of monocyte chemoattractant protein-1 (MCP-1), a chemokine that is controlled by the activation of central proinflammatory transcription factors, such as nuclear factor-kappa B (NF-kappaB). We found that human APC (2.5-10 micro g/ml) upregulated the amount of MCP-1-mRNA in human umbilical vein endothelial cells (HUVEC) and caused a time- and dose-dependent increase in MCP-1 protein production (p0.001 for APC 2.5 micro g/ml at 4 up to 24 h). In this cell culture model MCP-1 induced an improvement of cell migration and wound repair after injury to endothelial monolayers. After stimulation of MCP-1-mRNA-transcription with TNF-alpha (0.1-1 ng/ml), HUVEC's were washed and an inhibitor of gene transcription, Actinomycin D (1 micro g/ml), was added in the presence or absence of APC. HUVEC's receiving APC contained more MCP-1-mRNA than controls after one hour and up to eight hours suggesting an inhibitory effect of APC on MCP-1-mRNA degradation (with APC: 753 +/- 56 atto mol of MCP-1-mRNA per ml of cell lysate vs. 263 +/- 60 atto mol/ml without APC at t =4 h; p0.001). Electrophoretic mobility shift assays revealed that APC attenuated NF-kappaB DNA-binding capacity implying that NF-kappaB may not be involved in the upregulatory effect of APC on MCP-1 production. The ability of APC to upregulate the production of MCP-1, most likely by increasing the stability of MCP-1-mRNA rather than by transcriptional activation via NF-B, identifies a novel immunomodulatory pathway, by which APC may control the local inflammatory reaction, thereby initiating wound repair and modulating the extent of endothelial injury.