We monitored the number of intravascular platelet-leukocyte aggregates (PLAs) and thrombotic occlusions (TOs) by intravascular microscopy in the mesentery of rats receiving antiphospholipid (aPL) immunoglobulin G (IgG) purified from the sera of patients with antiphospholipid syndrome. aPL IgG had no procoagulant effect, but it caused rapid endothelial deposition of fibrinogen, followed by PLA and TO in rats receiving an intraperitoneal injection of bacterial lipopolysaccharide 3 hours before IgG infusion. Anti-␤2-glycoprotein I-depleted aPL IgG failed to induce PLAs and TOs. C3 and C9 colocalized with aPL IgG on the mesenteric vessels. The number of PLAs and TOs was markedly reduced in C6-deficient rats and in animals treated with anti-C5 miniantibody, suggesting the contribution of the terminal complement (C) complex to the aPL antibody-mediated intravascular thrombosis. In conclusion, our data indicate that antibodies to ␤2-glycoprotein I trigger coagulation subsequent to a priming proinflammatory factor and that the terminal C complex is the main mediator of the coagulation process. (Blood. 2005; 106:2340-2346)

Objectives To assess risk factors for a first thrombotic event in confirmed antiphospholipid (aPL) antibody carriers and to evaluate the efficacy of prophylactic treatments.Methods Inclusion criteria were age 18-65 years, no history of thrombosis and two consecutive positive aPL results.Demographic, laboratory and clinical parameters were collected at enrolment, once a year during the follow-up and at the time of the thrombotic event, whenever that occurred.Results 258 subjects were prospectively observed between October 2004 and October 2008. The mean±SD follow-up was 35.0±11.9 months (range 1-48). A first thrombotic event (9 venous, 4 arterial and 1 transient ischaemic attack) occurred in 14 subjects (5.4%, annual incidence rate 1.86%). Hypertension and lupus anticoagulant (LA) were significantly predictive of thrombosis (both at p<0.05) and thromboprophylaxis was significantly protective during high-risk periods (p<0.05) according to univariate analysis. Hypertension and LA were identified by multivariate logistic regression analysis as independent risk factors for thrombosis (HR 3.8, 95% CI 1.3 to 11.1, p<0.05, and HR 3.9, 95% CI 1.1 to 14, p<0.05, respectively).Conclusions Hypertension and LA are independent risk factors for thrombosis in aPL carriers. Thromboprophylaxis in these subjects should probably be limited to high-risk situations.

Recombinant osteoprotegerin (OPG) promoted the adhesion of both primary polymorphonuclear neutrophils (PMNs) and leukemic HL60 cells to endothelial cells. Leukocyte/endothelial cell adhesion was promoted by short (peak at 1 hour) preincubation of either endothelial cells or PMNs with OPG, and the peak of proadhesive activity was observed in the same range of OPG concentrations detected in the sera of patients affected by cardiovascular diseases. Although the cognate high-affinity ligands for OPG, membrane receptor activator of nuclear factor-B ligand (RANKL) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), were detected at significant levels on both PMNs and HL60 cells, they were not expressed on the surface of endothelial cells. However, preincubation of OPG with heparin abrogated its proadhesive activity, whereas pretreatment of endothelial cells with chondroitinase plus heparinases significantly decreased the proadhesive activity of OPG. Taken together, these findings suggest the involvement of both the ligand binding and the N-terminal heparin-binding domains of OPG in mediating its pro-adhesive activity. The relevance of these in vitro findings was underscored by in vivo experiments, in which the topical administration of recombinant OPG increased leukocyte rolling and adhesion to rat mesenteric postcapillary venules. Our data suggest that a pathological increase of OPG serum levels might play an important role in promoting leukocyte/endothelial cell adhesion. IntroductionOsteoprotegerin (OPG), a soluble member of the tumor necrosis factor (TNF) receptor superfamily, was originally characterized for its ability to suppress osteoclast formation. 1 OPG inhibits osteoclastogenesis by binding to receptor activator of nuclear factor-B (NF-B) ligand (RANKL), a member of the TNF superfamily of cytokines, and preventing the interaction of RANKL with its high-affinity transmembrane receptor (receptor activator of NF-B [RANK]). 2 It has been shown that in the low nanomolar range, the binding interaction of OPG and RANKL is 1:1 3 and that dimerization of OPG results from noncovalent interactions mediated by the death domains and, to a lesser extent, by a C-terminal heparinbinding region. OPG dimer formation is required for the mechanism of inhibition of the RANKL/RANK receptor interaction. 3 OPG can also interact with another member of the TNF superfamily, TNF-related apoptosis inducing ligand (TRAIL), 4 which can kill a variety of cancer cell types both in vivo and in vitro. 5 A role for OPG as a neutralizing receptor for TRAIL under physiological conditions has been questioned in early studies, but mounting evidence now suggests that the OPG/TRAIL interaction is biologically important, at least in in vitro culture systems. 6 OPG has been shown to act in a paracrine or autocrine manner by binding TRAIL and promoting the survival of prostate cancer cells, 7 breast cancer cells, 8 and multiple myeloma cells. 9 Moreover, when a rationally designed small molecule mimic of OPG was examined for associa...

The infrequent occurrence of septic shock in patients with inherited deficiencies of the terminal complement components experiencing meningococcal disease led us to suspect that the terminal complement complex is involved in vascular leakage. To this end, the permeabilizing effect of the cytolytically inactive soluble terminal complement complex (SC5b-9) was tested in a Transwell system measuring the amount of fluorescein-labeled BSA (FITC-BSA) leaked through a monolayer of endothelial cells. The complex caused increased permeability to FITC-BSA after 15 min as opposed to the prompt response to bradykinin (BK). The effect of SC5b-9 was partially reduced by HOE-140 or CV-3988, two selective antagonists of BK B2 and platelet-activating factor receptors, respectively, and was completely neutralized by the mixture of the two antagonists. Also, DX-88, a specific inhibitor of kallikrein, partially inhibited the activity of SC5b-9. The permeabilizing factor(s) released after 30 min of incubation of endothelial cells with SC5b-9 caused a prompt leakage of albumin like BK. Intravital microscopy confirmed both the extravasation of circulating FITC-BSA across mesenteric microvessels 15 min after topical application of SC5b-9 and the complete neutralization by the mixture of HOE-140 and CV-3988. SC5b-9 induced opening of interendothelial junctions in mesenteric endothelium documented by transmission electron microscopy.

Intravital microscopy was used to monitor leukocyte traffic across rat mesenteric postcapillary venules induced by the inactive terminal complement (C) complex (iTCC) topically applied to ileal mesentery. Leukocytes started rolling within 15 minutes from the administration of iTCC, and by 1 hour they adhered almost completely to the endothelium emigrating from the vessels in the next 3 hours. C5a caused a similar, though less marked, effect, whereas boiled iTCC was inactive, excluding the contribution of contaminating lipopolysaccharide. The complex stimulated the migration of polymorphonuclear neutrophils (PMNs) across endothelial cells (ECs) in a transwell system after a 4-hour incubation of ECs with iTCC added to the lower chamber of the transwell, whereas a 30-minute incubation was sufficient for C5a and interleukin (IL)-8 to induce the passage of PMNs. C5a was not responsible for the effect of iTCC because this complex had no chemotactic activity and contained too small an amount of C5a to account for the transen- IntroductionTerminal complement (C) complex (TCC) represents the end product of the C-activation cascade and is formed by the assembly of the 5 late components of the C system. The best-known function of this complex is to insert into the phospholipid bilayer of the cell target as membrane attack complex (MAC) causing cytolysis. However, evidence collected in the last 2 decades has convincingly proved that MAC in sublytic amounts is also able to induce noncytolytic effects on several target cells. 1,2 An early manifestation of cell activation induced by MAC is the rise in Ca ϩϩ caused by the rapid influx of these ions from an extracellular source and their cytoplasmic redistribution from intracellular stores. 1 The endothelium is a potential target of MAC that can be formed directly on the cell membrane as a result of C activation triggered by cell-bound antibodies. Alternatively, MAC starts assembling in the circulation and subsequently interacts with endothelial cells (ECs). Cells attacked by MAC may undergo cytolysis, but, more frequently, they release and express on their surfaces molecules involved in important biologic functions. Mobilization of P-selectin, 3 enhancement of tumor necrosis factor (TNF)-␣-induced expression of adhesion molecules, 4 release of chemokines, monocyte chemotactic protein-1 (MCP-1), and interleukin (IL)-8, 5 and platelet-activating factor 6 are examples of pro-inflammatory effects stimulated by MAC on ECs. Moreover, MAC triggers the coagulation process, stimulating ECs to express tissue factor 7 and to shed MAC-enriched vesicles that support the formation of a prothrombinase complex. 8 Finally, MAC has been shown to create interendothelial gaps, causing structural changes of the endothelium, 9 and it contributes to the modification of vascular tone by promoting the release of the vasodilator prostaglandin I 2 10 and the vasoconstrictor thromboxane A 2 from ECs. 11 These noncytotoxic effects on ECs require the complex to insert into the cytoplasmic membrane to signa...

Helicobacter pylori induces an acute inflammatory response followed by a chronic infection of the human gastric mucosa characterized by infiltration of neutrophils/polymorphonuclear cells (PMNs) and mononuclear cells. The H. pylori neutrophil-activating protein (HP-NAP) activates PMNs, monocytes, and mast cells, and promotes PMN adherence to the endothelium in vitro. By using intravital microscopy analysis of rat mesenteric venules exposed to HP-NAP, we demonstrated, for the first time in vivo, that HP-NAP efficiently crosses the endothelium and promotes a rapid PMN adhesion. This HP-NAP-induced adhesion depends on the acquisition of a high affinity state of β2 integrin on the plasma membrane of PMNs, and this conformational change requires a functional p38 MAPK. We also show that HP-NAP stimulates human PMNs to synthesize and release a number of chemokines, including CXCL8, CCL3, and CCL4. Collectively, these data strongly support a central role for HP-NAP in the inflammation process in vivo: indeed, HP-NAP not only recruits leukocytes from the vascular lumen, but also stimulates them to produce messengers that may contribute to the maintenance of the flogosis associated with the H. pylori infection.

The endothelial layer represents a continuous physical barrier that controls coagulation and allows selective passage of soluble molecules and circulating cells across the vessel wall into the tissue. The functional activity of the endothelial cells may be influenced by their interaction with components of the complement system. In this review we shall discuss the complex interplay that can be established between the endothelium and complement proteins or activation products. Endothelial cells may also secrete several complement components which contribute to the circulating pool. This process can be regulated by cytokines and other pro-inflammatory stimuli. In addition, complement activation products stimulate endothelial cells to acquire a pro-inflammatory and pro-coagulant status. Expression of regulatory molecules on the cell surface provides protection against an undesired attack by complement activation products. Unrestricted complement activation under pathological conditions may lead to structural and functional changes of the endothelium resulting in vascular disease.

Background Activation of bradykinin-mediated B2 receptor has been shown to play an important role in the onset of angioedema associated with C1 inhibitor deficiency. This finding has led to the development of novel therapeutic drugs such as the B2 receptor antagonist icatibant. However, it is unclear whether other receptors expressed on endothelial cells contribute to the release of kinins and vascular leakage in these patients. The recognition of their role may have obvious therapeutic implications. Objective Our aim was to investigate the involvement of B1 and gC1q receptors in in vitro and in vivo models of vascular leakage induced by plasma samples obtained from patients with C1 inhibitor deficiency. Methods The vascular leakage was evaluated in vitro on endothelial cells by a transwell model system and in vivo on rat mesentery microvessels by intravital microscopy. Results We observed that the attack phase plasma from C1 inhibitor–deficient patients caused a delayed fluorescein-labeled albumin leakage as opposed to the rapid effect of bradykinin, whereas remission plasma elicited a modest effect compared with control plasma. The plasma permeabilizing effect was prevented by blocking the gC1q receptor–high-molecular-weight kininogen interaction, was partially inhibited by B2 receptor or B1 receptor antagonists, and was totally prevented by the mixture of the 2 antagonists. Involvement of B1 receptor was supported by the finding that albumin leakage caused by attack phase plasma was enhanced by IL-1β and was markedly reduced by brefeldin A. Conclusion Our data suggest that both B1 receptor and gC1q receptor are involved in the vascular leakage induced by hereditary and acquired angioedema plasma.