Factor XII in coagulation,inflammation and beyond

Factor XII (FXII) is a protease that is mainly produced in the liver and circulates in plasma as a single chain zymogen. Following contact with negatively charged surfaces, FXII is converted into the two-chain active form, FXIIa. FXIIa initiates the intrinsic blood coagulation pathway via activation of factor XI. Furthermore, it converts plasma prekallikrein to kallikrein (PK), which reciprocally activates FXII and liberates bradykinin from high molecular weight kininogen. In addition, FXIIa initiates fibrinolysis via PK-mediated urokinase activation and activates the classical complement pathway. Even though the main function of FXII seems to relate to the activation of the intrinsic coagulation pathway and the kallikrein-kinin system, a growing body of evidence suggests that FXII may also directly regulate cellular responses. In this regard, it has been found that FXII/FXIIa induces the expression of inflammatory mediators, promotes cell proliferation, and enhances the migration of neutrophils and lung fibroblasts. In addition, it has been reported that genetic ablation of FXII protects against neuroinflammation, reduces the formation of atherosclerotic lesions in Apoe^−/− mice, improves wound healing, and inhibits postnatal angiogenesis. Although the aforementioned effects can be partially explained by the downstream products of FXII activation, the ability of FXII/FXIIa to directly regulate cellular responses has recently emerged as an alternative hypothesis. These direct cellular reactions to FXII/FXIIa will be discussed in the review.     

FXII promotes proteolytic processing of the LRP1 ectodomain

BackgroundFactor XII (FXII) is a serine protease that is involved in activation of the intrinsic blood coagulation, the kallikrein-kinin system and the complement cascade. Although the binding of FXII to the cell surface has been demonstrated, the consequence of this event for proteolytic processing of membrane-anchored proteins has never been described.MethodsThe effect of FXII on the proteolytic processing of the low-density lipoprotein receptor-related protein 1 (LRP1) ectodomain was tested in human primary lung fibroblasts (hLF), alveolar macrophages (hAM) and in human precision cut lung slices (hPCLS). The identity of generated LRP1 fragments was confirmed by MALDI-TOF-MS. Activity of FXII and gelatinases was measured by S-2302 hydrolysis and zymography, respectively.ResultsHere, we demonstrate a new function of FXII, namely its ability to process LRP1 extracellular domain. Incubation of hLF, hAM, or hPCLS with FXII resulted in the accumulation of LRP1 ectodomain fragments in conditioned media. This effect was independent of metalloproteases and required FXII proteolytic activity. Binding of FXII to hLF surface induced its conversion to FXIIa and protected FXIIa against inactivation by a broad spectrum of serine protease inhibitors. Preincubation of hLF with collagenase I impaired FXII activation and, in consequence, LRP1 cleavage. FXII-triggered LRP1 processing was associated with the accumulation of gelatinases (MMP-2 and MMP-9) in conditioned media.ConclusionsFXII controls LRP1 levels and function at the plasma membrane by modulating processing of its ectodomain.General significanceFXII-dependent proteolytic processing of LRP1 may exacerbate extracellular proteolysis and thus promote pathological tissue remodeling.     

Binding of activated Factor XII to endothelial cells affects its inactivation by the C1-esterase inhibitor.

It is well known that activated Factor XII (FXIIa) and kallikrein are rapidly inactivated in plasma as a result of reaction with endogenous inhibitors. The purpose of this may be to prevent uncontrolled deleterious spreading and activation of target zymogens. Both FXII and the complex plasma prekallikrein/high molecular mass kininogen become activated when they bind, in a Zn2+-dependent manner, to receptors on human umbilical vein endothelial cells (HUVEC). The C1-esterase inhibitor (C1-INH) is by far the most efficient inhibitor of FXIIa. In the present study it has been investigated whether binding of FXIIa to HUVEC might offer protection against inactivation by C1-INH. It appeared that the relative amidolytic activity of purified FXIIa bound to the surface of HUVEC decreased according to the concentration of C1-INH in medium; however, the decrease was smaller than that measured for inactivation of FXIIa in solution. The secondary rate constant for the inactivation was 3-10-fold lower for cell-bound than for soluble FXIIa. The inactivation was found to be caused by C1-INH binding to cell-bound FXIIa. Accordingly, the amidolytic activity of saturated amounts of cell-bound FXIIa was reduced in the presence of C1-INH and was theoretically nonexistent at physiological C1-INH concentrations. Amidolytic activity was, however, present on HUVEC incubated with plasma indicating that the endogenous C1-INH did not completely abolish the activity of FXIIa generated during the incubation period. This supports the hypothesis that binding to endothelial cells protects the activated FXII against inactivation by its major endogenous inhibitor. Hence, the function of FXII may be localized at cellular surfaces.     

Endothelial Cell Permeability during Hantavirus Infection Involves Factor XII-Dependent Increased Activation of the Kallikrein-Kinin System

Hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS) are diseases caused by hantavirus infections and are characterized by vascular leakage due to alterations of the endothelial barrier. Hantavirus-infected endothelial cells (EC) display no overt cytopathology; consequently, pathogenesis models have focused either on the influx of immune cells and release of cytokines or on increased degradation of the adherens junction protein, vascular endothelial (VE)-cadherin, due to hantavirus-mediated hypersensitization of EC to vascular endothelial growth factor (VEGF). To examine endothelial leakage in a relevant in vitro system, we co-cultured endothelial and vascular smooth muscle cells (vSMC) to generate capillary blood vessel-like structures. In contrast to results obtained in monolayers of cultured EC, we found that despite viral replication in both cell types as well as the presence of VEGF, infected in vitro vessels neither lost integrity nor displayed evidence of VE-cadherin degradation. Here, we present evidence for a novel mechanism of hantavirus-induced vascular leakage involving activation of the plasma kallikrein-kinin system (KKS). We show that incubation of factor XII (FXII), prekallikrein (PK), and high molecular weight kininogen (HK) plasma proteins with hantavirus-infected EC results in increased cleavage of HK, higher enzymatic activities of FXIIa/kallikrein (KAL) and increased liberation of bradykinin (BK). Measuring cell permeability in real-time using electric cell-substrate impedance sensing (ECIS), we identified dramatic increases in endothelial cell permeability after KKS activation and liberation of BK. Furthermore, the alterations in permeability could be prevented using inhibitors that directly block BK binding, the activity of FXIIa, or the activity of KAL. Lastly, FXII binding and autoactivation is increased on the surface of hantavirus-infected EC. These data are the first to demonstrate KKS activation during hantavirus infection and could have profound implications for treatment of hantavirus infections.     

Activated human platelets induce factor XIIa-mediated contact activation

Earlier studies have shown that isolated platelets in buffer systems can promote activation of FXII or amplify contact activation, in the presence of a negatively charge substance or material. Still proof is lacking that FXII is activated by platelets in a more physiological environment. In this study we investigate if activated platelets can induce FXII-mediated contact activation and whether this activation affects clot formation in human blood.Human platelets were activated with a thrombin receptor-activating peptide, SFLLRN-amide, in platelet-rich plasma or in whole blood. FXIIa and FXIa in complex with preferentially antithrombin (AT) and to some extent C1-inhibitor (C1INH) were generated in response to TRAP stimulation. This contact activation was independent of surface-mediated contact activation, tissue factor pathway or thrombin. In clotting whole blood FXIIa-AT and FXIa-AT complexes were specifically formed, demonstrating that AT is a potent inhibitor of FXIIa and FXIa generated by platelet activation. Contact activation proteins were analyzed by flow cytometry and FXII, FXI, high-molecular weight kininogen, and prekallikrein were detected on activated platelets. Using chromogenic assays, enzymatic activity of platelet-associated FXIIa, FXIa, and kallikrein were demonstrated. Inhibition of FXIIa in non-anticoagulated blood also prolonged the clotting time.We conclude that platelet activation triggers FXII-mediated contact activation on the surface and in the vicinity of activated platelets. This leads specifically to generation of FXIIa-AT and FXIa-AT complexes, and contributes to clot formation. Activated platelets may thereby constitute an intravascular locus for contact activation, which may explain the recently reported importance of FXII in thrombus formation.     

Fibrinolytic properties of activated FXII.

Activated factor XII (FXIIa), the initiator of the contact activation system, has been shown to activate plasminogen in a purified system. However, the quantitative role of FXIIa as a plasminogen activator in contact activation-dependent fibrinolysis in plasma is still unclear. In this study, the plasminogen activator (PA) activity of FXIIa was examined both in a purified system and in a dextran sulfate euglobulin fraction of plasma by measuring fibrinolysis in a fibrin microtiter plate assay. FXIIa was found to have low PA activity in a purified system. Dextran sulfate potentiated the PA activity of FXIIa about sixfold, but had no effect on the PA activity of smaller fragments of FXIIa, missing the binding domain for negatively charged surfaces. The addition of small amounts of factor XII (FXII) to FXII-deficient plasma induced a large increase in contact activation-dependent PA activity, as measured in a dextran sulfate euglobulin fraction, which may be ascribed to FXII-dependent activation of plasminogen activators like prekallikrein. When more FXII was added, PA activity continued to increase but to a lesser extent. In normal plasma, the addition of FXII also resulted in an increase of contact activation-dependent PA activity. These findings suggested a significant contribution of FXIIa as a direct plasminogen activator. Indeed, at least 20% of contact activation-dependent PA activity could be extracted from a dextran sulfate euglobulin fraction prepared from normal plasma by immunodepletion of FXIIa and therefore be ascribed to direct PA activity of FXIIa. PA activity of endogenous FXIIa immunoadsorped from plasma could only be detected in the presence of dextran sulfate. From these results it is concluded that FXIIa can contribute significantly to fibrinolysis as a plasminogen activator in the presence of a potentiating surface.     

Rapid and cooperative binding of factor XII to human umbilical vein endothelial cells.

When activated, factor XII (FXII) has been shown to play a role in a series of proteolytic cascades including systems as the fibrinolytic, the coagulation, the kallikrein-kinin and the complement. How FXII is activated in vivo remains poorly understood as the concentration and density of surface bound negative charges known to trigger the activation in vitro is far from sufficient in vivo. Specific binding of FXII to cellular receptors in the blood stream may, however, solve this problem which may be a question of inter molecular vicinity enhanced by binding to any surface. Here we report that the Zn(2+)-dependent binding of FXII to endothelial cells is rapid, saturable, specific and cooperative. Each endothelial cell from the human umbilical veins was found to bind (417 +/- 202) x 10(3) molecules of FXII with a Kd of (65 +/- 23) nM and a Hill coefficient of 2.1. The binding was inhibited by alpha-FXIIa but not by beta-FXIIa. The Kd for binding alpha-FXIIa was (50 +/- 27) nM. The rate of association was found to be 1.9 x 10(5) M(-1). min(-1). A confirmed inhibition by HK increased the Kd without affecting the maximal number of binding sites and the Hill coefficient. The concentration of HK in serum did not prevent binding of FXII/FXIIa to cells incubated with serum supplemented with Zn2+. The optimal concentration of Zn(2+) was 15 microM for binding factor XII/FXIIa whether purified or in serum.     

Differential binding of factor XII and activated factor XII to soluble and immobilized fibronectin--localization of the Hep-1/Fib-1 binding site for activated factor XII

Fibronectins (FNs) are dimeric glycoproteins that adopt a globular conformation when present in plasma and solution and an extended conformation in the extracellular matrix. Factor XII (FXII) is a zymogen of the proteolytically active FXIIa that plays a role in thrombus stabilization by enhancing clot formation and in inflammation by enhancing bradykinin formation. To investigate whether the extracellular matrix could play a role in these events, we have recently shown that FXIIa, but not FXII, binds to the extracellular matrix (ECM), and suggested that FN may be the target for the binding. Immunofluorescence microscopy has in the present investigation confirmed that FXIIa added to the ECM colocalizes with FN deposited during growth of human umbilical vein endothelial cells. The aim of the present study, therefore, was to further elucidate the interaction between FXIIa and FN by the use of a solid face binding assay. This showed, like the binding to the ECM, that FXIIa, but not FXII, binds in a Zn2+-independent manner to immobilized FN. The K(D) for the binding was 8.5 +/- 0.9 nM (n = 3). The binding was specific for the immobilized FN, as the binding could not be inhibited by soluble FN. Furthermore, soluble FN did not bind to immobilized FXIIa. However, soluble FN could bind to FXII, and this binding inhibited the surface-induced autoactivation of FXII and subsequent binding of the generated FXIIa to immobilized FN. The presence of FXII in an anti-FN immunoprecipitate of plasma indicated that some FXII in plasma circulates bound to FN. The binding of FXIIa to FN was inhibited by gelatine and fibrin but not by heparin, indicating that FXIIa binds to immobilized FN through the type I repeat modules. Accordingly, FXIIa was found to bind to immobilized fragments of FN containing the type I repeat modules in the N-terminal domain to which fibrin and gelatine bind.     

Regulation of blood coagulation by the protein C system.

Protein C is a plasma, vitamin K-dependent zymogen of a serine protease that can inhibit blood coagulation. Protein C is regulated by a series of reactions known as the protein C pathway. The importance of this pathway is seen in the occurrence of thrombosis in individuals with deficiencies in elements of the pathway like protein C and protein S. Work on several steps in this pathway has revealed that mechanisms involved in activation of protein C and the expression of its anticoagulant activity have features that allow for the expression of the anticoagulant activity away from sites in which procoagulant reactions occur, but not systemically. Thrombin, the principal procoagulant enzyme at the site of an injury, is converted to an anticoagulant enzyme at distant sites through its interaction with the endothelial cell protein thrombomodulin. Structural and functional studies have revealed the importance of several domain structures in the modulation of thrombin activity. Structural features of both activated protein C and its substrates (coagulation factors V and VIII) are such that they require the localization of enzyme and substrate on the surface of phosphatidyl serine containing membranes for optimum activity.     

FXII

The plasma protein FXII (Hageman factor) has been shown to be linked with the plasma defence systems of coagulation, fibrinolysis, kallikrein-kinin and complement. It can be activated by surface contact activation and in solution. Surface contact activation is a complex phenomenon involving negatively charged surfaces, FXII, high molecular weight kininogen and plasma kallikrein. Fluid-phase activation can be effected by a variety of serine proteases. In both types of activation the FXII zymogen is converted to active enzymes. FXII levels in plasma are low or undetectable in both inherited deficiencies and in a variety of clinical conditions. FXII levels can also be elevated in some clinical conditions. Although discovered as a clotting protein FXII appears to play an important role in the kallikrein-kinin and fibrinolytic systems and also has effects on cells. Recent studies suggest that therapeutic blockade of activation of FXII can be of benefit in certain clinical conditions.     

VCAM-1 activation of endothelial cell protein tyrosine phosphatase 1B

Lymphocytes migrate from the blood into tissue by binding to and migrating across endothelial cells. One of the endothelial cell adhesion molecules that mediate lymphocyte binding is VCAM-1. We have reported that binding to VCAM-1 activates endothelial cell NADPH oxidase for the generation of reactive oxygen species (ROS). The ROS oxidize and stimulate an increase in protein kinase C (PKC)alpha activity. Furthermore, these signals are required for VCAM-1-dependent lymphocyte migration. In this report, we identify a role for protein tyrosine phosphatase 1B (PTP1B) in the VCAM-1 signaling pathway. In primary cultures of endothelial cells and endothelial cell lines, Ab cross-linking of VCAM-1 stimulated an increase in serine phosphorylation of PTP1B, the active form of PTP1B. Ab cross-linking of VCAM-1 also increased activity of PTP1B. This activation of PTP1B was downstream of NADPH oxidase and PKCalpha in the VCAM-1 signaling pathway as determined with pharmacological inhibitors and antisense approaches. In addition, during VCAM-1 signaling, ROS did not oxidize endothelial cell PTP1B. Instead PTP1B was activated by serine phosphorylation. Importantly, inhibition of PTP1B activity blocked VCAM-1-dependent lymphocyte migration across endothelial cells. In summary, VCAM-1 activates endothelial cell NADPH oxidase to generate ROS, resulting in oxidative activation of PKCalpha and then serine phosphorylation of PTP1B. This PTP1B activity is necessary for VCAM-1-dependent transendothelial lymphocyte migration. These data show, for the first time, a function for PTP1B in VCAM-1-dependent lymphocyte migration.     

Heparan Sulfate Proteoglycans Mediate Factor XIIa Binding to the Cell Surface

Hageman factor (FXIIa) initiates the intrinsic coagulation pathway and triggers the kallikrein-kinin and the complement systems. In addition, it functions as a growth factor by expressing promitogenic activities toward several cell types. FXIIa binds to the cell surface via a number of structurally unrelated surface receptors; however, the underlying mechanisms are not yet fully understood. Here, we demonstrate that FXIIa utilizes cell membrane-bound glycosaminoglycans to interact with the cell surface of human lung fibroblasts (HLF). The combination of enzymatic, inhibitory, and overexpression approaches identified a heparan sulfate (HS) component of proteoglycans as an important determinant of the FXIIa binding capacity of HLF. Moreover, cell-free assays and competition experiments revealed preferential binding of FXIIa to HS and heparin over dextran sulfate, dermatan sulfate, and chondroitin sulfate A and C. Finally, we demonstrate that fibroblasts isolated from the lungs of the patients suffering from idiopathic pulmonary fibrosis (IPF) exhibit enhanced FXIIa binding capacity. Increased sulfation of HS resulting from elevated HS 6-O-sulfotransferase-1 expression in IPF HLF accounted, in part, for this phenomenon. Application of RNA interference technology and inhibitors of intracellular sulfation revealed the cooperative action of cell surface-associated HS and urokinase-type plasminogen activator receptor in the accumulation of FXIIa on the cell surface of IPF HLF. Moreover, FXIIa stimulated IPF HLF migration, which was abrogated by pretreatment of cells with heparinase I. Collectively, our study uncovers a novel role of HS-type glycosaminoglycans in a local accumulation of FXIIa on the cell membrane. The enhanced association of FXIIa with IPF HLF suggests its contribution to fibrogenesis.     

A Quantitative-Trait Locus in the Human Factor XII Gene Influences Both Plasma Factor XII Levels and Susceptibility to Thrombotic Disease

One approach to the identification of genetic loci that influence complex diseases is through the study of quantitative risk factors correlated with disease susceptibility. Factor XII (FXII) plasma levels, a related phenotype correlated with thrombosis, is such a risk factor. We conducted the first genomewide linkage screen to localize genes that influence variation in FXII levels. Two loci were detected: one on chromosome 5 and another on chromosome 10 (LOD scores 4.73 and 3.53, respectively). On chromosome 5, the peak LOD score occurred in the 5q33-5ter region, near the FXII gene. Addition of a 46C/T mutation in the FXII gene increased the multipoint LOD score to 10.21 (P=3.6 x 10(-12)). A bivariate linkage analysis of FXII activity and thrombosis further improved the linkage signal (LOD = 11.73) and provided strong evidence that this quantitative-trait locus (QTL) has a pleiotropic effect on the risk of thrombosis (P=.004). Linkage analysis conditional on 46C/T indicated that this mutation alone cannot explain the chromosome 5 signal, implying that other functional sites must exist. These results represent the first direct genetic evidence that a QTL in or near the FXII gene influences both FXII activity and susceptibility to thrombosis and suggest the presence of one or more still unknown functional variants in FXII.     

Sucrose Synthetase of Rice Grains and Potato Tubers

ABSTRACT

Human coagulation factor XII, the initiating factor in the intrinsic coagulation pathway, is critical for pathological thrombosis but not for hemostasis. Pharmacologic inhibition of factor XII is an attractive alternative in providing protection from pathologic thrombus formation while minimizing hemorrhagic risk. Large quantity of recombinant active factor XII is required for screening inhibitors and further research. In the present study, we designed and expressed the recombinant serine protease domain of factor XII in Pichia pastoris strain X-33, which is a eukaryotic expression model organism with low cost. The purification protocol was simplified and the protein yield was high (~20 mg/L medium). The purified serine protease domain of factor XII behaved homogeneously as a monomer, exhibited comparable activity with the human βFXIIa, and accelerated clot formation in human plasma. This study provides the groundwork for factor XII inhibitors screening and further research.     

Platelets promote coagulation factor XII-mediated proteolytic cascade systems in plasma

Blood coagulation factor XII (FXII, Hageman factor) is a plasma serine protease which is autoactivated following contact with negatively charged surfaces in a reaction involving plasma kallikrein and high-molecular-weight kininogen (contact phase activation). Active FXII has the ability to initiate blood clotting via the intrinsic pathway of coagulation and inflammatory reactions via the kallikrein-kinin system. Here we have determined FXII-mediated bradykinin formation and clotting in plasma. Western blotting analysis with specific antibodies against various parts of the contact factors revealed that limited activation of FXII is sufficient to promote plasma kallikrein activation, resulting in the conversion of high-molecular-weight kininogen and bradykinin generation. The presence of platelets significantly promoted FXII-initiated bradykinin formation. Similarly, in vitro clotting assays revealed that platelets critically promoted FXII-driven thrombin and fibrin formation. In summary, our data suggest that FXII-initiated protease cascades may proceed on platelet surfaces, with implications for inflammation and clotting.     

Dephosphorylation of endothelial nitric-oxide synthase by vascular endothelial growth factor. Implications for the vascular responses to cyclosporin A

The endothelial isoform of nitric-oxide synthase (eNOS) is a key determinant of vascular tone. eNOS, a Ca(2+)/camodulin-dependent enzyme, is also regulated by a variety of agonist-activated protein kinases, but the role and regulation of the protein phosphatase pathways involved in eNOS dephosphorylation are much less well understood. Treatment of endothelial cells with vascular endothelial growth factor (VEGF), a potent eNOS agonist, leads to the activation of calcineurin, a Ca(2+)/camodulin-dependent protein phosphatase. In these studies, we used a phosphorylation state-specific antibody to show that VEGF promotes dephosphorylation of eNOS at serine residue 116 in cultured endothelial cells. Cyclosporin A, an inhibitor of calcineurin, completely blocks VEGF-induced eNOS dephosphorylation; under identical conditions, cyclosporin A also inhibits VEGF-induced eNOS activation. VEGF-induced eNOS dephosphorylation shows an EC(50) of 2 ng/ml and is maximal 30 min after agonist addition. eNOS phosphorylation at serine 116 is completely blocked by the protein kinase C inhibitor calphostin but is blocked by neither wortmannin (an inhibitor of phosphatidylinositide 3-kinase) nor the MAP kinase pathway inhibitor U0126. A phosphorylation-deficient mutant of eNOS in which serine 116 is changed to an alanine residue (S116A) shows significantly enhanced enzyme activity compared with the wild-type enzyme. Taken together, these findings indicated that VEGF-induced eNOS dephosphorylation at serine 116 leads to enzyme activation. Cyclosporin A is widely used as an immunosuppressive drug for which hypertension is an important dose-limiting side effect. Our results suggest that cyclosporin A-induced hypertension may involve, at least in part, the attenuation of endothelium-derived NO production through a calcineurin-sensitive pathway regulating eNOS dephosphorylation.     

Expression,refolding, and activation of the catalytic domain of human blood coagulation factor XII

Human blood coagulation factor XII (FXII; 80 kDa) contains a C-terminal serine protease zymogen domain, which becomes activated upon contacting a negative surface. Activated FXII (alphaFXIIa) brings about reciprocal activation of FXII and kallikrein that by further hydrolysis produces the free catalytic domain (betaFXIIa; 28 kDa). Increased levels of alphaFXIIa are associated with coronary heart disease, sepsis, and diabetes. Biophysical investigation of the structural basis of activation, substrate specificity, and regulation of FXII requires an efficient bacterial system for producing the wild-type and mutant recombinant proteins. Here, the cDNA of the zymogen domain of FXII (betaFXII) was cloned into the pET-28a(+) vector and the plasmid was transformed into Escherichia coli strain BL21 (DE3) and overexpressed. The multi-disulfide, recombinant protein, His(6)-betaFXII (rbetaFXII), expressed as an inclusion body, was purified by means of a Ni(2+)-charged resin. The matrix-bound rbetaFXII was subjected to refolding with the glutathione redox system and activated by the in vivo activator, kallikrein. The active form, rbetaFXIIa, obtained in milligram quantities, exhibited similar structural and comparable functional properties relative to human betaFXIIa, as indicated by circular dichroism spectroscopy and kinetics of substrate hydrolysis. Thermodynamics of enzyme:inhibitor complex formation, including the expected 1:1 stoichiometry, was determined for rbetaFXIIa by isothermal calorimetric titration with a specific recombinant protein inhibitor, Cucurbita maxima trypsin inhibitor-V (rCMTI-V; 7kDa).     

Oxidized low-density lipoprotein increases cultured human endothelial cell tissue factor activity and reduces protein C activation

Increasing evidence suggests that the formation of oxidized low-density lipoprotein (Ox-LDL) in vivo is associated with the development of atherosclerotic vascular disease. We investigated the effects of Ox-LDL on two vascular endothelial cell coagulant properties, tissue factor expression, and protein C activation. The Ox-LDL increased human arterial and venous endothelial cell tissue factor activity, with 100 micrograms/ml of Ox-LDL increasing factor activity fourfold. Native LDL modified by incubation with cultured human arterial and venous endothelial cells also induced endothelial cell tissue factor activity. This modification was blocked by coincubation with the antioxidants, probucol or ascorbic acid. It was determined, based on inhibition by known scavenger receptor antagonists (fucoidin, dextran sulfate), that binding of Ox-LDL via the acetyl LDL (scavenger) receptor was partially responsible for the increase in tissue factor expression. Whereas endothelial cell tissue factor expression was increased by incubation with Ox-LDL, protein C activation was reduced approximately 80% by incubating cultured endothelial cells with Ox-LDL. The effect of Ox-LDL on protein C activation was not inhibited by antagonists to the scavenger receptor. These data indicating that an atherogenic lipoprotein can regulate key vascular coagulant activities provide an additional link between vascular disease and thrombosis.     

Insulin signaling in vascular endothelial cells: a key role for heterotrimeric G proteins revealed by siRNA-mediated Gbeta1 knockdown

Activation of insulin receptors stimulates the phosphoinositide 3-kinase (PI3-K)/Akt signaling pathway in vascular endothelial cells. Heterotrimeric G proteins appear to modulate some of the cellular responses that are initiated by receptor tyrosine kinases, but the roles of specific G protein subunits in signaling are less clearly defined. We found that insulin treatment of cultured bovine aortic endothelial cells (BAEC) activates the alpha isoform of PI3-K (PI3-Kalpha) and discovered that purified G protein Gbeta1gamma2 inhibits PI3-Kalpha enzyme activity. Transfection of BAEC with a duplex siRNA targeting bovine Gbeta1 leads to a 90% knockdown in Gbeta1 protein levels, with no effect on expression of other G protein subunits. siRNA-mediated Gbeta1 knockdown markedly and specifically potentiates insulin-dependent activation of kinase Akt, likely reflecting the removal of the inhibitory effect of Gbetagamma on PI3-Kalpha activity. Insulin-induced tyrosine phosphorylation of insulin receptors is unaffected by Gbeta1 siRNA. By contrast, Gbeta1 knockdown leads to a significant decrease in the level of serine phosphorylation of the insulin receptor substrate IRS-1. We explored the effects of siRNA on several serine/threonine protein kinases that have been implicated in insulin signaling. Gbeta1 siRNA significantly attenuates phosphorylation of the 70 kDa ribosomal protein S6 kinase (p70S6K) in the basal state and following insulin treatment. We also found that IGF-1-initiated activation of Akt is significantly enhanced after siRNA-mediated Gbeta1 knockdown, while IGF-1-induced p70S6K activation is markedly suppressed following transfection of Gbeta1 siRNA. We propose that Gbeta1 participates in the activation of p70S6K, which in turn promotes the serine phosphorylation and inhibition of IRS-1. Taken together, these studies suggest that Gbeta1 plays an important role in insulin and IGF-1 signaling in endothelial cells, both by inhibiting the activity of PI3-Kalpha and by stimulating pathways that lead to activation of protein kinase p70S6K and to the serine phosphorylation of IRS-1.     

The intrinsic apoptotic pathway is required for lipopolysaccharide-induced lung endothelial cell death

LPS has been implicated in the pathogenesis of endothelial cell death associated with Gram-negative bacterial sepsis. The binding of LPS to the TLR-4 on the surface of endothelial cells initiates the formation of a death-inducing signaling complex at the cell surface. The subsequent signaling pathways that result in apoptotic cell death remain unclear and may differ among endothelial cells in different organs. We sought to determine whether LPS and cycloheximide-induced cell death in human lung microvascular endothelial cells (HmVECs) was dependent upon activation of the intrinsic apoptotic pathway and the generation of reactive oxygen species. We found that cells overexpressing the anti-apoptotic protein Bcl-X(L) were resistant to LPS and cycloheximide-induced death and that the proapoptotic Bcl-2 protein Bid was cleaved following treatment with LPS. The importance of Bid was confirmed by protection of Bid-deficient (bid(-/-)) mice from LPS-induced lung injury. Neither HmVECs treated with the combined superoxide dismutase/catalase mimetic EUK-134 nor HmVECs depleted of mitochondrial DNA (rho(0) cells) were protected against LPS and cycloheximide-induced death. We conclude that LPS and cycloheximide-induced death in HmVECs requires the intrinsic cell death pathway, but not the generation of reactive oxygen species.