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In addition to cholesterol-lowering properties, statins exhibit lipid-independent immunomodulatory, anti-inflammatory actions. However, high concentrations are typically required to induce these effects in vitro, raising questions concerning therapeutic relevance. We present evidence that endothelial cell sensitivity to statins depends upon shear stress. Using heme oxygenase-1 expression as a model, we demonstrate differential heme oxygenase-1 induction by atorvastatin in atheroresistant compared with atheroprone sites of the murine aorta. In vitro, exposure of human endothelial cells to laminar shear stress significantly reduced the statin concentration required to induce heme oxygenase-1 and protect against H(2)O(2)-mediated injury. Synergy was observed between laminar shear stress and atorvastatin, resulting in optimal expression of heme oxygenase-1 and resistance to oxidative stress, a response inhibited by heme oxygenase-1 small interfering RNA. Moreover, treatment of laminar shear stress-exposed endothelial cells resulted in a significant fall in intracellular cholesterol. Mechanistically, synergy required Akt phosphorylation, activation of Kruppel-like factor 2, NF-E2-related factor-2 (Nrf2), increased nitric-oxide synthase activity, and enhanced HO-1 mRNA stability. In contrast, heme oxygenase-1 induction by atorvastatin in endothelial cells exposed to oscillatory flow was markedly attenuated. We have identified a novel relationship between laminar shear stress and statins, demonstrating that atorvastatin-mediated heme oxygenase-1-dependent antioxidant effects are laminar shear stress-dependent, proving the principle that biomechanical signaling contributes significantly to endothelial responsiveness to pharmacological agents. Our findings suggest statin pleiotropy may be suboptimal at disturbed flow atherosusceptible sites, emphasizing the need for more specific therapeutic agents, such as those targeting Kruppel-like factor 2 or Nrf2.

Complement activation may predispose to vascular injury and atherogenesis. The atheroprotective actions of unidirectional laminar shear stress led us to explore its influence on endothelial cell expression of complement inhibitory proteins CD59 and decay-accelerating factor. Human umbilical vein and aortic endothelial cells were exposed to laminar shear stress (12 dynes/cm(2)) or disturbed flow (+/- 5 dynes/cm(2) at 1Hz) in a parallel plate flow chamber. Laminar shear induced a flow rate-dependent increase in steady-state CD59 mRNA, reaching 4-fold at 12 dynes/cm(2). Following 24-48 h of laminar shear stress, cell surface expression of CD59 was up-regulated by 100%, whereas decay-accelerating factor expression was unchanged. The increase in CD59 following laminar shear was functionally significant, reducing C9 deposition and complement-mediated lysis of flow-conditioned endothelial cells by 50%. Although CD59 induction was independent of PI3-K, ERK1/2 and nitric oxide, an RNA interference approach demonstrated dependence upon an ERK5/KLF2 signaling pathway. In contrast to laminar shear stress, disturbed flow failed to induce endothelial cell CD59 protein expression. Likewise, CD59 expression on vascular endothelium was significantly higher in atheroresistant regions of the murine aorta exposed to unidirectional laminar shear stress, when compared with atheroprone areas exposed to disturbed flow. We propose that up-regulation of CD59 via ERK5/KLF2 activation leads to endothelial resistance to complement-mediated injury and protects from atherogenesis in regions of laminar shear stress.

Background: Heme oxygenase-1 (HO-1), by exerting anti-inflammatory, antiproliferative, antiapoptotic and antioxidant effects in the vasculature, protects against atherosclerosis and post-transplant vasculopathy. We noted the overlap between the effects of HO-1 and those attributed to 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins). This led to an investigation of the role of HO-1 in statin-mediated cytoprotection in primary human endothelial cells (ECs), and the ability of Kruppel-like factor 2 (KLF2) to regulate HO-1 function., Methods/results: Treatment of human umbilical vein and aortic ECs with atorvastatin significantly upregulated HO-1 promoter activity, mRNA expression and protein expression, increasing HO-1 enzymatic activity as shown by raised intracellular bilirubin IXalpha. This effect was indirect, dependent upon inhibition of HMG-CoA reductase and geranylgeranylation, and independent of nitric oxide or changes in mRNA stability. Atorvastatin protected ECs against the generation of reactive oxygen species and H(2)O(2)-induced injury. HO-1 inhibition, with small interfering RNA (siRNA) or zinc protoporphyrin IX, abrogated atorvastatin-mediated cytoprotection. Atorvastatin upregulated KLF2 expression, whereas KLF2 siRNA attenuated statin-induced HO-1 and its associated antioxidant cytoprotective effects. Iron chelation, adenoviral-mediated overexpression of ferritin or supplementation of culture media with biliverdin reversed the inhibitory effects of HO-1 and KLF2 siRNA, suggesting that bile pigments and ferritin mediate the antioxidant actions of statin-induced HO-1., Conclusions: We have identified a novel link between KLF2 and HO-1 in human vascular ECs, demonstrating that atorvastatin-mediated HO-1 upregulation, and its associated antioxidant effect, is KLF2-dependent. The relationship between KLF2 and HO-1 is likely to represent an important component of the vasculoprotective profile of statins.

Endothelial cell apoptosis is associated with vascular injury and predisposes to atherogenesis. Endothelial cells express anti-apoptotic genes including Bcl-2, Bcl-XL and survivin, which also contribute to angiogenesis and vascular remodeling. We report a central role for protein kinase Cepsilon (PKCepsilon) in the regulation of Bcl-2 expression and cytoprotection of human vascular endothelium against apoptosis. Using myristoylated inhibitory peptides, a predominant role for PKCepsilon in vascular endothelial growth factor-mediated endothelial resistance to apoptosis was revealed. Immunoblotting of endothelial cells infected with an adenovirus expressing a constitutively active form of PKCepsilon (Adv-PKCepsilon-CA) or control Adv-beta-galactosidase demonstrated a 3-fold, PKCepsilon-dependent increase in Bcl-2 expression, with no significant change in Bcl-XL, Bad, Bak, or Bax. The induction of Bcl-2 inhibited apoptosis induced by serum starvation or etoposide, and PKCepsilon activation attenuated etoposide-induced caspase-3 cleavage. The functional role of Bcl-2 was confirmed with Bcl-2 antagonist HA-14-1. Inhibition of phosphoinositide 3-kinase attenuated vascular endothelial growth factor-induced protection against apoptosis, and this was rescued by overexpression of constitutively active PKCepsilon, suggesting PKCepsilon acts downstream of phosphoinositide 3-kinase. Co-immunoprecipitation studies demonstrated a physical interaction between PKCepsilon and Akt, which resulted in formation of a signaling complex, leading to optimal induction of Bcl-2. This study reveals a pivotal role for PKCepsilon in endothelial cell cytoprotection against apoptosis. We demonstrate that PKCepsilon forms a signaling complex and acts co-operatively with Akt to protect human vascular endothelial cells against apoptosis through induction of the anti-apoptotic protein Bcl-2 and inhibition of caspase-3 cleavage.

The in vitro isolation, propagation and study of endothelial cells (EC) is an invaluable means by which the function of the vascular endothelium in physiology and patho-physiology can be explored. In recent years heterogeneity between large and small vessel EC, between arteries and veins, and between microvascular EC derived from different organs has become increasingly apparent. This has led to the development of protocols for the isolation of these different EC. In addition, the data emerging on vascular EC function in gene-targeted mice has highlighted the need for reliable methods of isolation of murine EC. This chapter describes methods for the isolation, characterization and culture of macro- and microvascular EC from a variety of species and introduces simple approaches to investigating their surface antigen expression.

Objective: Thrombin is generated in significant quantities during cardiopulmonary bypass and mediates adverse events, such as platelet aggregation and proinflammatory responses, through activation of the high-affinity thrombin receptor protease-activated receptor 1, which is expressed on platelets and endothelium. Thus antagonism of protease-activated receptor 1 might have broad therapeutic significance. Aprotinin, used clinically to reduce transfusion requirements and the inflammatory response to bypass, has been shown to inhibit protease-activated receptor 1 on platelets in vitro and in vivo. Here we have examined whether aprotinin inhibits endothelial protease-activated receptor 1 activation and resulting proinflammatory responses induced by thrombin., Methods: Protease-activated receptor 1 expression and function were examined in cultured human umbilical vein endothelial cells after treatment with alpha-thrombin at 0.02 to 0.15 U/mL in the presence or absence of aprotinin (200-1600 kallikrein inhibitory units/mL). Protease-activated receptor 1 activation was assessed by using an antibody, SPAN-12, which detects only the unactivated receptor, and thrombin-mediated calcium fluxes. Other thrombin-dependent inflammatory pathways investigated were phosphorylation of the p42/44 mitogen-activated protein kinase, upregulation of the early growth response 1 transcription factor, and production of the proinflammatory cytokine interleukin 6., Results: Pretreatment of cultured endothelial cells with aprotinin significantly spared protease-activated receptor 1 receptor cleavage (P < .0001) and abrogated calcium fluxes caused by thrombin. Aprotinin inhibited intracellular signaling through p42/44 mitogen-activated protein kinase (P < .05) and early growth response 1 transcription factor (P < .05), as well as interleukin 6 secretion caused by thrombin (P < .005)., Conclusions: This study demonstrates that endothelial cell activation by thrombin and downstream inflammatory responses can be inhibited by aprotinin in vitro through blockade of protease-activated receptor 1. Our results provide a new molecular basis to help explain the anti-inflammatory properties of aprotinin reported clinically.

Thrombin, an important mediator of thrombosis and inflammation, may also enhance vascular cytoprotection. Thus thrombin induces expression of the complement-inhibitory protein decay-accelerating factor (DAF) in human umbilical vein endothelial cells (HUVECs), thus increasing protection against complement-mediated injury. Using PKC isozyme-specific peptide antagonists and adenoviral constructs, we have shown in the present study that PKC-epsilon is the primary isozyme involved in DAF induction by thrombin. Experiments with proteinase-activated receptor-1 (PAR1) and PAR2 activating peptides (APs) showed that DAF expression induced by PAR1-AP was PKC-alpha-dependent; in contrast, PAR2-AP induction of DAF required activation of PKC-epsilon. PAR1-AP and PAR2-AP in combination exerted an additive effect on DAF protein expression, which was equivalent to that observed with thrombin alone. These data implied a specific role for PAR2 in DAF induction, which was supported by the observation that upregulation of endothelial cell (EC) PAR2-enhanced DAF induction by thrombin. ERK1/2, p38, and JNK MAPK were also involved in thrombin-induced DAF upregulation, with evidence of interdependence between ERK1/2 and JNK. A role for transactivation of PAR2 by PAR1 was suggested by partial inhibition of thrombin-induced DAF expression by the PAR1 signaling antagonists BMS-200261 and SCH79797, whereas inhibition of thrombin-induced cleavage of PAR1 by specific MAbs or hirudin completely abrogated the response. Together, these data imply that the predominant pathway for thrombin-induced DAF expression involves transactivation of PAR2 by PAR1 and signaling via PKC-epsilon/MAPK. This may represent an important, novel pathway for endothelial cytoprotection during inflammation and angiogenesis and suggests that PAR2 may play a central role in some thrombin-induced responses.

Objectives: Vascular cell adhesion molecule-1 (VCAM-1) is a cell surface adhesion molecule involved in the recruitment of leukocytes to endothelial cells on arterial walls during the pathogenesis of atherosclerosis. The soluble ectodomain of VCAM-1 (sVCAM-1) is proteolytically released from the cell surface into the circulation, a process which is up-regulated in patients with cardiovascular or inflammatory disease. Here we investigate mechanisms involved in sVCAM-1 generation in response to cytokine stimulation., Methods: VCAM-1 ectodomain release into the conditioned media of MCEC-1 murine endothelial cells and cells grown from primary aortic explants from timp3-/- mice and wild-type littermates was measured by sandwich ELISA and Western blot after stimulation with tumor necrosis factor alpha (TNFalpha), interleukin-1beta (IL-1beta), or the phorbol ester PMA. Protease expression was inhibited (knocked down) with siRNA and validated using real-time PCR., Results: Proinflammatory cytokines IL-1beta and TNFalpha up-regulated VCAM-1 ectodomain release from the MCEC-1 cells, and this was dependant on p38 and mitogen-activated protein kinases (MAP kinases) and inhibited by the matrix metalloproteinase (MMP) inhibitor BB94 and tissue inhibitor of metalloproteinase (TIMP)-3, but not TIMP-1 or TIMP-2. Timp-3-/- cells exhibited greater VCAM-1 ectodomain release following cytokine stimulation than TIMP-3-expressing cells. Additionally, cytokine stimulation of MCEC-1 cells was shown to cause down-regulation of TIMP-3 expression. Knockdown of the metalloproteinase ADAM17, but not ADAM10 or ADAM12, gene expression reduced cytokine-stimulated VCAM-1 shedding., Conclusions: TIMP-3 regulates the release of sVCAM-1 from cytokine-stimulated endothelial cells, which is mediated by ADAM17.

Decay-accelerating factor (DAF), a membrane-bound complement regulatory protein, is up-regulated on endothelial cells (ECs) following treatment with vascular endothelial growth factor (VEGF), providing enhanced protection from complement-mediated injury. We explored the signaling pathways involved in this response. Incubation of human umbilical vein ECs with VEGF induced a 3-fold increase in DAF expression. Inhibition by flk-1 kinase inhibitor SU1498 and failure of placental growth factor (PlGF) to up-regulate DAF confirmed the role of VEGF-R2. The response was also blocked by pretreatment with phospholipase C-gamma (PLCgamma) inhibitor U71322 and protein kinase C (PKC) antagonist GF109203X. In contrast, no effect was seen with nitric oxide synthase inhibitor N(G)-monomethyl-l-arginine (l-NMMA). Use of PKC agonists and isozyme-specific pseudosubstrate peptide antagonists suggested a role for PKCalpha and -epsilon in VEGF-mediated DAF up-regulation. This was confirmed by transfection of ECs with PKCalpha and -epsilon dominant-negative constructs, which in combination completely abrogated induction of DAF by VEGF. In contrast, LY290042, a phosphoinositide 3-kinase (PI3K) inhibitor, significantly augmented DAF expression, suggesting a negative regulatory role for phosphoinositide 3-kinase. The widely used immunosuppressive drug cyclosporin A (CsA) inhibited DAF induction by VEGF in a dose-dependent manner. The VEGF-induced DAF expression was functionally effective, significantly reducing complement-mediated EC lysis, and this cytoprotective effect was reversed by CsA. These data provide evidence for a VEGF-R2-, phospholipase C-gamma-, and PKCalpha/epsilon-mediated cytoprotective pathway in ECs. This may represent an important mechanism for the maintenance of vascular integrity during chronic inflammation involving complement activation. Moreover, inhibition of this pathway by CsA may play a role in CsA-mediated vascular injury.

We have shown that human endothelial cells (EC) are protected against complement-mediated injury by the inducible expression of decay-accelerating factor (DAF). To understand further the importance of DAF regulation, we characterized EC DAF expression on murine EC in vitro and in vivo using a model of glomerulonephritis. Flow cytometry using the monoclonal antibody (mAb) Riko-3 [binds transmembrane- and glycosylphosphatidylinositol (GPI)-anchored DAF], mAb Riko-4 (binds GPI-anchored DAF) and reverse transcription-polymerase chain reaction (RT-PCR), demonstrated that murine EC DAF is GPI-anchored. Tumour necrosis factor-alpha (TNF-alpha) increased EC DAF expression, detectable at 6 hr and maximal at 24-48 hr poststimulation. DAF upregulation required increased steady-state DAF mRNA and protein synthesis. In contrast, no increased expression of the murine complement receptor-related protein-Y (Crry) was seen with TNF-alpha. DAF upregulation was mediated via a protein kinase C (PKC)alpha, phosphoinositide-3 kinase (PI-3 kinase), p38 mitogen-activated protein kinase (MAPK) and nuclear factor-kappaB (NF-kappaB)-dependent pathway. The increased DAF was functionally relevant, resulting in a marked reduction in C3 deposition following complement activation. In a nephrotoxic nephritis model, DAF expression on glomerular capillaries was significantly increased 2 hr after the induction of disease. The demonstration of DAF upregulation above constitutive levels suggests that this may be important in the maintenance of vascular integrity during inflammation, when the risk of complement-mediated injury is increased. The mouse represents a suitable model for the study of novel therapeutic approaches by which vascular endothelium may be conditioned against complement-mediated injury.

Complement-mediated vascular injury is important in the pathophysiology of atherosclerosis and myocardial infarction. Because recent evidence shows that statins have beneficial effects on endothelial cell (EC) function independent of lipid lowering, we explored the hypothesis that statins modulate vascular EC resistance to complement through the upregulation of complement-inhibitory proteins. Human umbilical vein and aortic ECs were treated with atorvastatin or simvastatin, and decay-accelerating factor (DAF), membrane cofactor protein, and CD59 expression was measured by flow cytometry. A dose-dependent increase in DAF expression of up to 4-fold was seen 24 to 48 hours after treatment. Statin-induced upregulation of DAF required increased steady-state mRNA and de novo protein synthesis. L-Mevalonate and geranylgeranyl pyrophosphate reversed the effect, confirming the role of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibition and suggesting that constitutive DAF expression is negatively regulated by geranylgeranylation. Neither farnesyl pyrophosphate nor squalene inhibited statin-induced DAF expression, suggesting that the effect is independent of cholesterol lowering. Statin-induced DAF upregulation was mediated by the activation of protein kinase Calpha and inhibition of RhoA and was independent of phosphatidylinositol-3 kinase and NO activity. The increased DAF expression was functionally effective, resulting in significant reduction of C3 deposition and complement-mediated lysis of antibody-coated ECs. These observations provide evidence for a novel cytoprotective action of statins on vascular endothelium that is independent of the effect on lipids and results in enhanced protection against complement-mediated injury. Modulation of complement regulatory protein expression may contribute to the early beneficial effects of statins in reducing the morbidity and mortality associated with atherosclerosis.

The complement-regulatory protein decay-accelerating factor (DAF) can be upregulated on endothelial cells (EC) by protein kinase C (PKC)-dependent and -independent pathways. We hypothesized that basic fibroblast growth factor (bFGF) might induce EC DAF expression, providing a cytoprotective mechanism for angiogenic neovessels against complement-mediated injury. Incubation of umbilical vein, aortic, and dermal EC with bFGF or vascular endothelial growth factor (VEGF) significantly increased DAF expression. Growth factor-induced EC proliferation was inhibited by PKC antagonists. In contrast, although PKC antagonists inhibited VEGF-induced DAF expression, bFGF-induced DAF was unaffected. Investigation of mitogen-activated kinase (MAPK) pathways also revealed differences, with bFGF-induced DAF dependent on p44/42 and p38 MAPK and VEGF requiring activation of p38 MAPK alone. Upregulation of DAF by bFGF was functionally relevant, reducing C3 deposition on EC after complement activation by 60% and resulting in marked reduction in complement-mediated EC lysis. bFGF and VEGF were synergistic in terms of DAF expression, resulting in enhanced cytoprotection. These observations reveal parallel PKC-dependent and -independent pathways regulating complement activation during angiogenesis. Further elucidation of these pathways may provide important insights into innate cytoprotective mechanisms in endothelium.

Although mouse endothelial cells (EC) may advance our understanding of endothelial function, primary EC remain difficult to isolate. We have established a murine cardiac endothelial cell line (MCEC-1) from transgenic mice harboring a temperature-sensitive simian virus 40 large TAg gene (tsA58 TAg) under H-2K(b) class I promoter control. MCEC-1 cells were characterized by their ability to form tubes, Griffonia simplicifolia isolectin B4 binding, and CD31, intercellular adhesion molecule (ICAM)-2, and endoglin expression. MCEC-1 cells proliferated rapidly under permissive conditions [33 degrees C with interferon (IFN)-gamma], where the T antigen is active and transcription is activated by the presence of IFN-gamma, whereas under nonpermissive conditions (38 degrees C without IFN-gamma) proliferation was reduced by 30-fold and the EC showed enhanced proliferation in response to growth factors. Expression of E- and P-selectin, ICAM-1, and vascular cell adhesion molecule-1 was upregulated by tumor necrosis factor-alpha and interleukin-1 beta, and MCEC-1 cells, in contrast to primary EC, were amenable to transfection by lipofection. This novel line will allow further study of the role of the endothelium in cardiovascular disease. Moreover, this technique will allow EC to be readily obtained from genetically modified mice backcrossed with H-2K(b)-tsA58 mice.

Background: Cardiopulmonary bypass surgery is often accompanied by a systemic inflammatory response, which can lead to postoperative complications in high-risk patients. This is mediated in part through a systemic rise in inflammatory cytokine levels and the sequestration of leukocytes within organs. Aprotinin has previously been shown to exert an anti-inflammatory effect by preventing the capacity of leukocytes to transmigrate through vascular endothelium. Here we have focused on whether aprotinin has an effect on endothelial cell activation and adhesion molecule expression in response to tumor necrosis factor-alpha, particularly with reference to whether aprotinin inhibits tumor necrosis factor-stimulated neutrophil transendothelial migration., Methods and Results: Intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and E-selectin expression was studied in tumor necrosis factor-alpha-activated human umbilical vein endothelial cells in the presence of aprotinin at 200, 800, and 1600 kIU/mL. Aprotinin inhibited tumor necrosis factor-alpha-stimulated expression of intercellular adhesion molecule-1 (P =.019 at 1600 kIU/mL) and vascular cell adhesion molecule-1 (P =.003 at 1600 kIU/mL) but not E-selectin. Similar results were obtained in the dermal microvascular endothelial cell line, HMEC-1, which exhibited diminished intercellular adhesion molecule-1 expression in the presence of aprotinin (P =.040 at 800 kIU/mL and P <.001 at 1600 kIU/mL). Aprotinin also significantly inhibited neutrophil transmigration across tumor necrosis factor-alpha-activated human umbilical vein endothelial cells (P =.046 at 1600 kIU/mL)., Conclusions: We have demonstrated that aprotinin inhibits intercellular adhesion molecule-1 and vascular cell adhesion molecule-1, but not E-selectin, expression on tumor necrosis factor-alpha-activated endothelial cells and that transendothelial migration by neutrophils is also specifically suppressed under these conditions. Our results indicate that endothelial cells can be specifically targeted by aprotinin, therefore adding to our understanding of the anti-inflammatory mechanism of action of aprotinin during cardiopulmonary bypass.

Objective: Decay-accelerating factor (DAF) is a widely expressed, multifunctional cell surface protein involved in complement regulation and cell signaling. Previous studies have demonstrated that endothelial cell (EC) DAF is up-regulated by tumor necrosis factor alpha and inhibits complement binding. Because vascular endothelial growth factor (VEGF) is cytoprotective to endothelium and is expressed at sites of chronic inflammation, we hypothesized that VEGF may induce DAF expression during inflammatory angiogenesis., Methods: Human umbilical vein and dermal microvascular EC were isolated using routine procedures, and the regulation and function of DAF, as well as other complement-regulatory proteins (membrane cofactor protein and CD59), were analyzed following stimulation with VEGF., Results: Incubation of large- or small-vessel EC with VEGF led to increased expression of DAF, with maximal expression after 48-72 hours of stimulation. This effect depended on the activation of protein kinase C (PKC) and required increased steady-state messenger RNA levels and de novo protein synthesis. Although VEGF-induced EC proliferation was inhibited by both p38 and p42/44 mitogen-activated protein kinase (MAPK) antagonists, DAF up-regulation in response to VEGF was only sensitive to inhibition of p38 MAPK. VEGF-stimulated EC showed a 60% reduction in C3 deposition following complement activation, and this resulted in a marked reduction in complement-mediated EC lysis. These protective effects were abolished by anti-DAF monoclonal antibody 1H4., Conclusion: This study confirms the importance of PKC for the regulation of DAF expression by EC and reveals VEGF to be a physiologic agonist for this pathway. The up-regulation of DAF expression by VEGF may represent an important mechanism for the protection of EC from complement-mediated injury during angiogenesis in inflammatory rheumatic diseases.

The isolation and long-term culture of murine endothelial cells (ECs) has often proven a difficult task. In this paper we describe a quick, efficient protocol for the isolation of microvascular endothelial cells from murine tissues. Murine lung or heart are mechanically minced and enzymatically digested with collagenase and trypsin. The single cell suspension obtained is then incubated with an anti-CD31 antibody, anti-CD105 antibody and with biotinylated isolectin B-4. Pure EC populations are finally obtained by magnetic bead separation using rat anti-mouse Ig- and streptavidin-conjugated microbeads. EC cultures are subsequently expanded and characterised. The surface molecule expression by the primary cultures of murine EC obtained from lung and heart tissue is analysed and compared to that of a murine endothelioma and of primary cultures of murine renal tubular epithelial cells. The phenotype and morphology of these cultures remain stable over 10-15 passages in culture, and no overgrowth of contaminating cells of non-endothelial origin is observed at any stage.

There is increasing evidence for functional crosstalk between inflammatory and thrombotic pathways in inflammatory vascular diseases such as atherosclerosis and vasculitis. Thus, complement activation on the endothelial cell (EC) surface during inflammation may generate thrombin via the synthesis of tissue factor. We explored the hypothesis that thrombin induces EC expression of the complement-regulatory proteins decay-accelerating factor (DAF), membrane cofactor protein (MCP), and CD59 and that this maintains vascular integrity during coagulation associated with complement activation. Thrombin increased DAF expression on the surface of ECs by 4-fold in a dose- and time-dependent manner as measured by flow cytometry. DAF up-regulation was first detectable at 6 hours and maximal 24 hours poststimulation, whereas no up-regulation of CD59 or MCP was seen. Thrombin-induced expression required increased DAF messenger RNA and de novo protein synthesis. The response depended on activation of protease-activated receptor 1 (PAR1) and was inhibited by pharmacologic antagonists of protein kinase C (PKC), p38 and p42/44 mitogen-activated protein kinase, and nuclear factor-kappa B. The increased DAF expression was functionally relevant because it significantly reduced C3 deposition and complement-mediated EC lysis. Thus, thrombin-generated at inflammatory sites in response to complement activation-is a physiologic agonist for the PKC-dependent pathway of DAF regulation, thereby providing a negative feedback loop protecting against thrombosis in inflammation. (Blood. 2000;96:2784-2792)

Background: Aprotinin is a serine protease inhibitor used extensively in cardiac operations to reduce postoperative bleeding. It has also been used in trials aimed at reducing the systemic inflammatory response to cardiopulmonary bypass. It remains unclear whether the anti-inflammatory action of aprotinin is related to its general ability to suppress leukocyte activation or whether aprotinin can exercise effects during the leukocyte-endothelial cell adhesion cascade., Methods: We used intravital microscopy to study the 3 main stages of the adhesion cascade (leukocyte rolling, firm adhesion, and extravasation) within the mesenteric microcirculation of rats. This in vivo technique allows leukocyte recruitment to be viewed directly through the transparent mesentery of anesthetized animals., Results: Aprotinin, given by continuous infusion at a clinically relevant dose, exerted no effect on the rolling or firm adhesion responses toward local chemoattractant N -formyl-methyl-leucyl-phenylalanine but significantly inhibited extravasation of leukocytes (73% at 40 minutes, P =.04) into surrounding tissues. In parallel in vitro experiments, aprotinin (used at 200, 800, and 1600 kIU/mL) dose dependently inhibited neutrophil transmigration through cultured endothelial cells in response to 3 different chemoattractants: N -formyl-methyl-leucyl-phenylalanine (P <.001 at 800 and 1600 kIU/mL), interleukin 8 (P <.05 at 200 kIU/mL and P <.001 at 800 and 1600 kIU/mL), and platelet-activating factor (P <.05 at 1600 kIU/mL)., Conclusions: Our studies have therefore revealed a novel anti-inflammatory mechanism of aprotinin operating at the level of leukocyte extravasation. These findings may be relevant in the prevention of systemic inflammation after cardiopulmonary bypass through the use of protease inhibitors.

In vivo cell-mediated immune reactions are characterized by mixtures of monocytes and T cells. The purpose of this study was to investigate the role of monocytes on T-cell migration and induction of endothelial adhesion molecules. The in vitro model consisted of adding peripheral blood mononuclear cells (PBMC), T cells or mixtures of monocytes and T cells, to endothelial cells on a porous membrane and using flow cytometry to distinguish between the monocyte and lymphocyte components. PBMC and PBMC supernatants were highly potent at upregulating intercellular adhesion molecule-1 (ICAM-1) and inducing expression of vascular cell adhesion molecule-1 (VCAM-1) and E-selectin. Induction by supernatants was inhibited by antibodies to tumour necrosis factor-alpha (TNF-alpha) and interleukin-1 (IL)-1 beta. Using monocyte-enriched populations, as few as one monocyte to 100 endothelial cells was sufficient to upregulate adhesion molecules. Fixed monocytes also induced adhesion molecules and expressed surface-bound cytokines. In contrast, highly purified unstimulated T cells were not found to induce adhesion molecules at 4, 6, 24 or 48 hr of coculture. Purified T cells showed low-level migration through resting (VCAM-1 negative) endothelium, which was approximately doubled by addition of small numbers of monocytes or TNF-alpha. In conclusion, monocytes, via cell surface or released cytokines play an essential role in allowing large-scale recruitment of T cells to inflammatory sites in vivo.