Cytokine-activated Endothelial Cells Research Articles

Targeting gene delivery to activated vascular endothelium using anti E/P-Selectin antibody linked to PAMAM dendrimers

Introduction The adhesion molecules P- and E-selectin are expressed on activated endothelial cells, and are good targets for gene therapy aimed at inflammatory disease. We have therefore investigated the potential of targeting PAMAM dendrimers, a non viral vector system, to cells expressing P/E-selectin using a monoclonal antibody that recognises these molecules. Materials and Methods We used biotin and avidin to cross-link anti E/P-Selectin monoclonal antibody to pre-formed Superfect-DNA complexes that were then used to transfect reporter genes to CHO cells expressing E-Selectin, cytokine-activated primary Human Saphenous Vein Endothelial Cells (HSVEC) and whole vein segments. Results The use of the anti E/P-Selectin antibody increased the transfection efficiency in CHO-E cells, activated HSVEC and saphenous vein segments ex vivo. We also showed that the antibody improved the binding of the complexes onto cells as well as the internalisation kinetics. Discussion We demonstrate here that by attaching antibodies onto PAMAM dendrimers the efficiency of transfection can be significantly improved in cells or tissues expressing the receptor. This technology has potential in the treatment of cardiovascular disease by gene therapy but can also be used with different antibodies to target other diseased cells or tissues.

Dances with leukocytes: how tetraspanin-enriched microdomains assemble to form endothelial adhesive platforms

Rather than just providing an unstructured adhesive surface for leukocytes, cytokine-activated endothelial cells assemble preexisting tetraspanin-enriched microdomains to form endothelial adhesive platforms (EAPs) and endothelial docking structures. In this issue of the Journal of Cell Biology, Barreiro et al. (Barreiro, O., M. Zamai, M. Yáñez-Mó, E. Tejera, P. López-Romero, P.N. Monk, E. Gratton, V.R. Caiolfa, and F. Sánchez-Madrid. 2008. J. Cell Biol. 183:527–542) show how the immunoglobulin superfamily adhesion molecules intercellular adhesion molecule (ICAM)–1 and vascular cell adhesion molecule (VCAM)–1 form nanoclusters with the tetraspanins CD9 and CD151 in a physiologically relevant system. Furthermore, convincing biochemical data suggest that these structures are distinct from lipid rafts.

Intercellular adhesion molecule-1 (ICAM-1) expression and soluble ICAM-1 (sICAM-1) production by cytokine-activated human aortic endothelial cells: a possible role for ICAM-1 and sICAM-1 in atherosclerotic aortic aneurysms.

The interactions of inflammatory cells, cytokines, and cell adhesion molecules (CAM) may be important in the pathogenesis of vascular diseases such as abdominal aortic aneurysms (AAA), in which inflammation plays a role. The aim of this study was to investigate the pathogenic role of ICAM-1, a molecule involved in leucocyte-endothelial interactions, in vascular inflammation. ELISA of human explant culture supernatants revealed a four-fold increase in sICAM-1 production by AAA (n = 9) versus normal (n = 8) aortic explants. Human aortic endothelial cell (hAEC) culture was used for further studies as an in vitro model for aortic inflammatory conditions. Tumour necrosis factor-alpha (TNF-alpha) or IL-1 beta treatment of hAEC resulted in an up to 1.8-fold significant increase in sICAM-1 production compared with resting cells. In addition, the expression of ICAM-1 on cytokine-stimulated versus resting hAEC was measured by radioimmunoassay. TNF-alpha significantly induced ICAM-1 expression on these cells. These results suggest that different forms of ICAM-1, present on or released by the activated aortic endothelium, may be involved in leucocyte adhesion to and migration into the vessel wall.

Role of the Protein C Pathway in the Extraintestinal Thrombosis Associated With Murine Colitis

Chronic inflammatory bowel diseases (IBD) are associated with an increased risk for thromboembolism. Although thrombosis is known to contribute to the morbidity and mortality of patients with IBD, the underlying mechanisms that contribute to the genesis of a hypercoagulable state during intestinal inflammation remain poorly defined. The objective of this study was to determine whether the protein C pathway contributes to the enhanced extraintestinal thrombosis that is associated with dextran sodium sulfate (DSS)-induced colitis in mice. Microvascular thrombosis was induced in cremaster muscle microvessels of normal and colitic mice using a light/dye injury model. DSS colitis enhanced thrombus formation in cremaster arterioles of wild-type mice. The DSS-induced thrombosis response was greatly attenuated in transgenic mice over expressing the endothelial protein C receptor. Activated protein C (APC), administered to colitic WT mice immediately prior to photoactivation, also afforded protection against thrombosis, and an anti-APC antibody enhanced thrombus formation. These findings indicate that elevated APC levels, derived from either endogenous or exogenous sources, confer protection against the extraintestinal thrombosis that accompanies colonic inflammation.

Clustering endothelial E-selectin in clathrin-coated pits and lipid rafts enhances leukocyte adhesion under flow

During inflammation, E-selectin expressed on cytokine-activated endothelial cells mediates leukocyte rolling under flow. E-selectin undergoes endocytosis and may associate with lipid rafts. We asked whether distribution of E-selectin in membrane domains affects its functions. E-selectin was internalized in transfected CHO cells or cytokine-activated human umbilical vein endothelial cells (HUVECs). Confocal microscopy demonstrated colocalization of E-selectin with alpha-adaptin, a clathrin-associated protein. Deleting the cytoplasmic domain of E-selectin or disrupting clathrin-coated pits with hypertonic medium blocked internalization of E-selectin, reduced colocalization of E-selectin with alpha-adaptin, and inhibited E-selectin-mediated neutrophil rolling under flow. Unlike CHO cells, HUVECs expressed a small percentage of E-selectin in lipid rafts. Even fewer neutrophils rolled on E-selectin in HUVECs treated with hypertonic medium and with methyl-beta-cyclodextrin, which disrupts lipid rafts. These data demonstrate that E-selectin clusters in both clathrin-coated pits and lipid rafts of endothelial cells but is internalized in clathrin-coated pits. Distribution in both domains markedly enhances E-selectin's ability to mediate leukocyte rolling under flow.

Role of the renin angiotensin system in TNF-α and Shiga-toxin-induced tissue factor expression

Current evidence implicates a prothrombotic state in the development of Shiga-toxin (Stx)-mediated hemolytic uremic syndrome (HUS). We recently reported that Stx modulates procoagulant activity by enhancing functional tissue factor (TF) activity on cytokine-activated human glomerular endothelial cells (HGECs). Since angiotensin II (Ang II), the key effector of the renin angiotensin system (RAS), has been shown to increase TF expression in vascular tissue, we examined the possible involvement of Ang II in TF expression in HGECs. HGECs were exposed to tumor necrosis factor (TNF)-alpha +/- Stx-1 +/- Ang II. Exogenous Ang II significantly increased TF activity and TF mRNA in TNF-alpha- +/- Stx-1-activated HGECs. This increase was mediated via Ang II type I receptor (AT(1)R), as losartan, an AT(1)R inhibitor, attenuated Ang-II-induced TF activity. To study the effect of endogenous Ang II in TF expression by TNF-alpha +/- Stx-1, HGECs were incubated with losartan or an AT(2)R inhibitor (PD 123319) or an angiotensin-converting enzyme inhibitor (enalapril). Losartan but not PD 123319 decreased TF activity induced by TNF-alpha +/- Stx-1 (P < 0.05). Enalapril, also, dose dependently, downregulated TF expression in HGECs exposed to TNF-alpha +/- Stx-1 (P < 0.05). AT(1)R mRNA was upregulated in TNF-alpha- +/- Stx-1-activated HGECs (P < 0.05). These data indicate that TF expression in TNF-alpha- and Stx-1-activated HGECs is enhanced by exogenous Ang II and that endogenous Ang II production may be upregulated by TNF-alpha +/- Stx-1. Hence, local RAS activation may be important in the development of the thrombotic microangiopathy observed in HUS.

Endothelial PI 3-kinase activity regulates lymphocyte diapedesis

Lymphocyte recruitment to sites of inflammation involves a bidirectional series of cues between the endothelial cell (EC) and the leukocyte that culminate in lymphocyte migration into the tissue. Remodeling of the EC F-actin cytoskeleton has been observed after leukocyte adhesion, but the signals to the EC remain poorly defined. We studied the dependence of peripheral blood lymphocyte transendothelial migration (TEM) through an EC monolayer in vitro on EC phosphatidylinositol 3-kinase (PI 3-kinase) activity. Lymphocytes were perfused over cytokine-activated EC using a parallel-plate laminar flow chamber. Inhibition of EC PI 3-kinase activity using LY-294002 or wortmannin decreased lymphocyte TEM (48 +/- 6 or 34 +/- 7%, respectively, vs. control; mean +/- SE; P < 0.05). Similarly, EC knockdown of the p85alpha regulatory subunit of PI 3-kinase decreased lymphocyte transmigration. Treatment of EC with jasplakinolide to inhibit EC F-actin remodeling also decreased lymphocyte TEM to 24 +/- 10% vs. control (P < 0.05). EC PI 3-kinase inhibition did not change the strength of lymphocyte adhesion to the EC or formation of the EC "docking structure" after intercellular adhesion molecule-1 ligation, whereas this was inhibited by jasplakinolide treatment. A similar fraction of lymphocytes migrated on control or LY-294002-treated EC and localized to interendothelial junctions. However, lymphocytes failed to extend processes below the level of vascular endothelial (VE)-cadherin on LY-294002-treated EC. Together these observations indicate that EC PI 3-kinase activity and F-actin remodeling are required during lymphocyte diapedesis and identify a PI 3-kinase-dependent step following initial separation of the VE-cadherin barrier.

Antiendothelial cell antibodies mediate enhanced leukocyte adhesion to cytokine-activated endothelial cells through a novel mechanism requiring cooperation between FcγRIIa and CXCR1/2

AbstractAntiendothelial cell antibodies (AECAs) are commonly detectable in diseases associated with vascular injury, including systemic lupus erythematosus (SLE), systemic sclerosis, Takayasu arteritis, Wegener granulomatosis, Behçet syndrome, and transplant arteriosclerosis. Here, we explore the hypothesis that these antibodies might augment polymorphonuclear leukocyte (PMN) adhesion to endothelium in inflammation. Initially, we established that a mouse IgG mAb bound to endothelial cells (ECs) significantly increased PMN adhesion to cytokine-stimulated endothelium in an FcγRIIa-dependent manner. Neutralizing antibodies, and adenoviral transduction of resting ECs, demonstrated that the combination of E-selectin, CXCR1/2, and β2 integrins is both necessary and sufficient for this process. We observed an identical mechanism using AECA IgG isolated directly from patients with SLE. Assembled immune complexes also enhanced PMN adhesion to endothelium, but, in contrast to adhesion because of AECAs, this process did not require CXCR1/2, was not inhibited by pertussis toxin, and was FcγRIIIb rather than FcγRIIa dependent. These data are the first to demonstrate separate nonredundant FcγRIIa and FcγRIIIb-mediated mechanisms by which EC-bound monomeric IgG and assembled immune complexes amplify leukocyte adhesion under dynamic conditions. Furthermore, the observation that FcγRIIa and CXCR1/2 cooperate to enhance PMN recruitment in the presence of AECAs suggests a mechanism whereby AECAs may augment tissue injury during inflammatory responses.

Regulation of NF-κB-dependent Gene Expression by the POU Domain Transcription Factor Oct-1

Maintenance of the cells of the vessel wall in a quiescent state is an important aspect of normal vascular physiology. Transcriptional repressors are widely believed to regulate this process, yet the exact factors involved and the mechanism of repression are not known. Here, we report that the POU domain transcription factor Oct-1 represses the expression of E-selectin and vascular cell adhesion molecule (VCAM-1), two cytokine-inducible, NF-kappaB-dependent endothelial-leukocyte adhesion molecules that participate in the leukocyte recruitment phase of the inflammatory response. Co-transfection and microinjection studies demonstrate that Oct-1 blocks tumor necrosis factor alpha-stimulated E-selectin and VCAM-1 expression. Gene expression arrays indicate that control of tumor necrosis factor alpha-induced, NF-kappaB-dependent gene expression by Oct-1 is promoter-specific. A DNA-binding mutant of Oct-1 represses NF-kappaB-dependent reporter gene expression. Biochemically, Oct-1 interacts with p65, suggesting that Oct-1 is involved in the regulation of NF-kappaB transactivation function. NF-kappaB-dependent gene expression is more pronounced in Oct-1-deficient than in wild-type murine embryonic fibroblasts, and reintroduction of human Oct-1 abolishes these differences. Finally, the cytokine interleukin-6 induces Oct-1 gene expression, providing a biologically relevant means by which NF-kappaB-dependent gene expression can be selectively reverted by Oct-1 to quiescent levels.

CD16+ monocytes produce IL-6, CCL2, and matrix metalloproteinase-9 upon interaction with CX3CL1-expressing endothelial cells

The CD16+ subset of peripheral blood monocytes (Mo) is expanded dramatically during inflammatory conditions including sepsis, HIV-1 infection, and cancer. CD16+ express high levels of CX3CR1, which mediates arrest onto CX3CL1-expressing endothelial cells (EC) under flow conditions. In contrast, attachment of CD16- Mo onto cytokine-activated EC is independent of CX3CL1. Here, we investigate the ability of CD16+ and CD16- Mo to produce proinflammatory cytokines upon interaction with CX3CL1-expressing HUVEC. We demonstrate that CD16+ but not CD16- Mo produce high levels of IL-6, CCL2, and matrix metalloproteinase (MMP)-9 when cocultured with TNF/IFN-gamma-activated HUVEC or nonactivated HUVEC expressing CX3CL1. Furthermore, supernatants from Mo cocultured with cytokine-activated HUVEC induce neuronal death in vitro. These results suggest that membrane-bound CX3CL1 stimulates production of IL-6, CCL2, and MMP-9 by CD16+ Mo, likely via engagement of CX3CR1. Thus, expansion of CD16+ Mo and their accumulation onto CX3CL1-expressing EC may result in recruitment of Mo and T cell subsets at sites of inflammation in response to CCL2, IL-6-induced cell activation and/or differentiation, and MMP-9-mediated vascular and tissue injury.

Cytokine-induced monocyte adhesion to endothelial cells involves platelet-activating factor: Suppression by conjugated linoleic acid

Monocyte–endothelium interaction is key to many acute and chronic inflammatory diseases. We have investigated the factors regulating monocyte attachment to cytokine-activated human umbilical vein endothelial cells (HUVEC) and the modulatory effect of the polyunsaturated fatty acid (PUFA), conjugated linoleic acid (CLA) in this process. Both TNF-α and IL-1β induced HUVEC platelet-activating factor (PAF) production and PAF was required for subsequent firm THP-1 monocyte adhesion since it was inhibited by both PAF receptor antagonists (BN-52021 or CV-6209) and a PAF synthesis inhibitor (sanguinarine). CLA inhibited the binding of both THP-1 and isolated human peripheral blood monocytes to HUVEC by up to 40% with the CLA t10,c12 isomer suppressing adhesion dose-dependently. Investigation into the mechanism involved demonstrated that with IL-1β, VCAM-1 and ICAM-1 levels and pro-inflammatory cytokine expression were largely unaffected by CLA. Through the use of PAF receptor antagonists and PAF synthesis inhibitors, CLA was shown to inhibit cytokine-induced binding by suppressing PAF production. Direct assay of PAF levels confirmed this result. We conclude that endothelial-generated PAF plays a central role in cytokine-induced monocyte adherence to endothelium and that the anti-inflammatory action of PUFAs such as CLA in suppressing monocyte–endothelial interaction is mediated through attenuation of pro-inflammatory phospholipids such as PAF.

Human Microvascular Endothelial Cell Activation by IL-1 and TNF-α Stimulates the Adhesion and Transendothelial Migration of Circulating Human CD14+ Monocytes That Develop With RANKL Into Functional Osteoclasts

Circulating pre-OCs may be recruited to locally inflamed sites through specific interactions with activated microvasculature. We found that HMVECs stimulated the adhesion and TEM of circulating pre-OCs, in an ICAM-1- and CD44-dependent manner, leading to greater RANKL-induced OC formation and bone pit resorption. Inflammation is critical for healing processes but causes severe tissue destruction when chronic. Local osteoclast (OC) formation and bone resorption may increase at inflammatory sites through multiple mechanisms, including direct stimulation by inflamed microvasculature of circulating OC precursor (pre-OC) migration through a blood vessel barrier into bone or joint tissue. How this might occur is not yet well understood. Cytokine-activated human microvascular endothelial cell (HMVEC) monolayers, with or without IL-1 and TNF-alpha preactivation (24 h), were incubated in adhesion (1-3 h) or porous transwell transendothelial migration (TEM; 3 h) assays with human peripheral blood mononuclear cells (hPBMCs) or CD14+ monocyte or CD14- lymphocyte subsets. The number of cells that adhered or transmigrated, and their ability to thereafter develop with macrophage-colony stimulating factor (M-CSF) + RANKL into bone pit-resorbing OCs, were analyzed. Immunostaining and neutralizing antibodies to key cell adhesion molecules were used to determine their potential involvement in stimulated CD14+ monocyte TEM. M-CSF + RANKL caused OC and bone pit formation only from hPBMCs and CD14+ cells but not CD14- cells. Adhesion of hPBMCs or CD14+ cells but not CD14- cells was stimulated by cytokine preactivation of HMVECs and led to the full capture of all circulating pre-OCs capable of developing into OCs. Cytokine-preactivated HMVECs also promoted the postadhesion TEM of hPBMCs and CD14+ populations, resulting in markedly greater OC formation and bone pit resorption by transmigrated cells. Immunodetectable vascular cell adhesion molecule (VCAM-1), intercellular adhesion molecule (ICAM-1), and CD44 levels increased on cytokine-treated HMVEC surfaces, and neutralizing antibodies to ICAM-1 or CD44, but not VCAM-1 or platelet endothelial cell adhesion molecule (PECAM-1), inhibited stimulated CD14+ cell TEM through activated HMVECs. This is the first demonstration that cytokine-activated HMVECs efficiently capture and promote the TEM of circulating pre-OCs capable of differentiating into bone-resorbing OCs. Thus, direct pre-OC recruitment by activated microvasculature at inflammatory sites may significantly contribute to normal OC bone remodeling during fracture healing or exacerbate pathological bone loss in various chronic inflammatory disorders.

HIV-1 Tat Regulates Endothelial Cell Cycle Progression via Activation of the Ras/ERK MAPK Signaling Pathway

Tat, the transactivator of HIV-1 gene expression, is released by acutely HIV-1-infected T-cells and promotes adhesion, migration, and growth of inflammatory cytokine-activated endothelial and Kaposi's sarcoma cells. It has been previously demonstrated that these effects of Tat are due to its ability to bind through its arginine-glycine-aspartic (RGD) region to the alpha5beta1 and alphavbeta3 integrins. However, the signaling pathways linking Tat to the regulation of cellular functions are incompletely understood. Here, we report that Tat ligation on human endothelial cells results in the activation of the small GTPases Ras and Rac and the mitogen-activated protein kinase ERK, specifically through its RGD region. In addition, we demonstrated that Tat activation of Ras, but not of Rac, induces ERK phosphorylation. We also found that the receptor proximal events accompanying Tat-induced Ras activation are mediated by tyrosine phosphorylation of Shc and recruitment of Grb2. Moreover, Tat enabled endothelial cells to progress through the G1 phase in response to bFGF, and the process is linked to ERK activation. Taken together, these data provide novel evidence about the ability of Tat to activate the Ras-ERK cascade which may be relevant for endothelial cell proliferation and for Kaposi's sarcoma progression.

Inhibition of E-selectin-mediated leukocyte adhesion by volatile anesthetics in a static condition

Leukocyte recruitment from blood vessels to inflamed tissues is the central step in the process of inflammation. This may cause damage of the inflamed tissues in the case of severe inflammatory conditions such as ischemia reperfusion or graft rejection. Adhesion molecules, such as E-selectin, are induced on activated endothelium and play an important role in this process. Volatile anesthetics protect tissues or organs in such conditions, and inhibition of leukocyte adhesion by anesthetics has been implicated. However, little is known about how the anesthetics act on individual adhesion molecules. We examined the effects of volatile anesthetics on E-selectin mediated leukocyte adhesion in a static condition using HL-60 cells, a granulocyte cell line, and E-selectin-coated plates as well as cytokine-activated human umbilical vein endothelial cells (HUVEC). The adhesion assay was carried out by overlaying fluorescence-labeled HL-60 cells on E-selectin-coated plates or cytokine-activated HUVEC. E-selectin in the coated plates or activated HUVEC were quantified by enzyme-linked immunosorbent assay. E-selectin in the activated HUVEC was analyzed by immunoblot. Isoflurane and sevoflurane concentration-dependently suppressed adhesion of HL-60 cells to E-selectin-coated plates. Although isoflurane did not change the amount of expression, or the molecular weight of E-selectin in the activated HUVEC, it significantly suppressed HL-60 cell adhesion to activated HUVEC. Volatile anesthetics suppress E-selectin-mediated cell adhesion in a static condition without changing the expression of E-selectin. A role for E-selectin in the organ protection by volatile anesthetics is suggested.

Vascular endothelium does not activate CD4+ direct allorecognition in graft rejection.

Expression of MHC class II by donor-derived APCs has been shown to be important for allograft rejection. It remains controversial, however, whether nonhemopoietic cells, such as vascular endothelium, possess Ag-presenting capacity to activate alloreactive CD4(+) T lymphocytes. This issue is important in transplantation, because, unlike hemopoietic APCs, allogeneic vascular endothelium remains present for the life of the organ. In this study we report that cytokine-activated vascular endothelial cells are poor APCs for allogeneic CD4(+) T lymphocytes in vitro and in vivo despite surface expression of MHC class II. Our in vitro observations were extended to an in vivo model of allograft rejection. We have separated the allostimulatory capacity of endothelium from that of hemopoietic APCs by using bone marrow chimeras. Hearts that express MHC class II on hemopoietic APCs are acutely rejected in a mean of 7 days regardless of the expression of MHC class II on graft endothelium. Alternatively, hearts that lack MHC class II on hemopoietic APCs are acutely rejected at a significantly delayed tempo regardless of the expression of MHC class II on graft endothelium. Our data suggest that vascular endothelium does not play an important role in CD4(+) direct allorecognition and thus does not contribute to the vigor of acute rejection.

Interactions of selectins with PSGL-1 and other ligands.

The selectins are type I membrane glycoproteins that mediate adhesion of leukocytes and platelets to vascular surfaces (McEver 2001; Vestweber and Blanks 1999). L-selectin is expressed on most leukocytes. E-selectin is expressed on cytokine-activated endothelial cells. P-selectin is rapidly redistributed from membranes of secretory granules to the surfaces of activated platelets and endothelial cells. Each selectin has a membrane-distal C-type lectin domain, followed by an epidermal growth factor (EGF)-like motif, a series of consensus repeats, a transmembrane domain, and a short cytoplasmic tail. P- and E-selectin bind primarily to ligands on leukocytes, and P-selectin also interacts with ligands on platelets and some endothelial cells. L-selectin binds to ligands on endothelial cells of high endothelial venules (HEV) in lymph nodes and on other leukocytes.

T‐cell responses in atopic eczema

T‐cell responses in atopic eczema

Regulation of zinc homeostasis by inducible NO synthase-derived NO: nuclear metallothionein translocation and intranuclear Zn2+ release.

Zn2+ is critical for the functional and structural integrity of cells and contributes to a number of important processes including gene expression. It has been shown that NO exogenously applied via NO donors resulting in nitrosative stress leads to cytoplasmic Zn2+ release from the zinc storing protein metallothionein (MT) and probably other proteins that complex Zn2+ via cysteine thiols. We show here that, in cytokine-activated murine aortic endothelial cells, NO derived from the inducible NO synthase (iNOS) induces a transient nuclear release of Zn2+. This nuclear Zn2+ release depends on the presence of MT as shown by the lack of this effect in activated endothelial cells from MT-deficient mice and temporally correlates with nuclear MT translocation. Data also show that NO is an essential but not sufficient signal for MT-mediated Zn2+ trafficking from the cytoplasm into the nucleus. In addition, we found that, endogenously via iNOS, synthesized NO increases the constitutive mRNA expression of both MT-1 and MT-2 genes and that nitrosative stress exogenously applied via an NO donor increases constitutive MT mRNA expression via intracellular Zn2+ release. In conclusion, we here provide evidence for a signaling mechanism based on iNOS-derived NO through the regulation of intracellular Zn2+ trafficking and homeostasis.

Critical role of L-arginine in endothelial cell survival during oxidative stress.

Oxidative damage of vascular endothelium represents an important initiation step in the development of atherosclerosis. Recently, we reported about protection of inducible nitric oxide synthase (iNOS)-derived high-output NO in endothelial cells. Because iNOS activity critically depends on the availability of its substrate l-arginine, the present study aims at elucidating iNOS-mediated effects on H2O2-induced apoptosis of cytokine-activated rat aortic endothelial cells (AECs) subject to medium l-arginine concentrations. In cytokine-activated AECs, iNOS activity was found to be half-maximal at 60 micromol/L arginine, which represents the medium serum level in rats but also in humans. Maximal activity is seen at and above 200 micromol/L arginine. Activated cells grown in the absence of arginine with minimal iNOS activity are highly sensitive toward H2O2-induced apoptosis, and increases in medium arginine concentrations result in increased cell survival. Moreover, competition experiments show that iNOS activity is completely dependent on cationic amino acid transporter-mediated arginine uptake. We also find that the arginine-dependent protection includes inhibition of endothelial lipid peroxidation and increases in the expression of vasoprotective stress response genes. Our data demonstrate that arginine concentrations corresponding to physiological serum levels do not allow for optimal endothelial iNOS activity. Thus, decreases in systemic arginine concentrations, or locally within atherosclerotic plaques, will impair the endothelial iNOS-mediated stress response and will significantly increase the risk of endothelial dysfunction.

Interaction of endothelial cells and neutrophils in vitro: kinetics of thrombomodulin, intercellular adhesion molecule-1 (ICAM-1), E-selectin, and vascular cell adhesion molecule-1 (VCAM-1): implications for the relevance as serological disease activity markers in vasculitides.

Recently markers of endothelial cell activation or injury gained increasing interest as serological parameters of disease activation in vasculitides. Among these, soluble serum thrombomodulin, ICAM-1, VCAM-1 and E-selectin are of particular interest. However, only thrombomodulin showed the expected close correlation. The objective of this study was to investigate in vitro the kinetics of these endothelial cell receptors after interaction of unstimulated or cytokine-activated polymorphonuclear neutrophils (PMN) and endothelial cells in order to find evidence explaining these different clinical findings. Over the time period of up to 48 h of incubation the kinetics of thrombomodulin, ICAM-1, E-selectin, and VCAM-1 levels in the supernatant of endothelial cells in co-culture with neutrophils were determined in vitro by ELISA under basal and partially cytokine-activated (tumour necrosis factor-alpha) conditions. Increased levels of ICAM-1, E-selectin and VCAM-1 were already found due to cytokine activation of endothelial cells alone. This increase was augmented after coincubation with neutrophils. In contrast, a significant increase of thrombomodulin in the supernatant was only found due to cell injury after cell-cell interaction of cytokine-activated endothelial cells with neutrophils. In conclusion, this in vitro model of the kinetics of soluble endothelial cell receptors after cell-cell interaction of cytokine-activated PMN and endothelial cells underlines the advantage of thrombomodulin in contrast to the adhesion molecules as a marker of endothelial damage. Therefore, soluble thrombomodulin seems to be a promising, valuable serological disease activity marker in vasculitides.