Henri H. Versteeg

Infliximab but not etanercept induces apoptosis in lamina propria T-lymphocytes from patients with Crohn’s disease

BACKGROUND & AIMS Steroid-refractory Crohns disease responds to therapy with the chimeric anti-tumor necrosis factor (TNF)-alpha antibody infliximab. Etanercept, a recombinant TNF receptor/immunoglobulin G fusion protein, is highly effective in rheumatoid arthritis but not in Crohns disease. Because both infliximab and etanercept are TNF-alpha-neutralizing drugs, we investigated the differences in TNF-alpha-neutralizing capacity and human lymphocyte binding and apoptosis-inducing capacity of both molecules. METHODS We used a nuclear factor kappaB reporter assay and a cytotoxicity bioassay to study TNF-alpha neutralization by infliximab and etanercept. Lymphocyte binding and apoptosis-inducing capacity was investigated using fluorescence-activated cell sorter analysis, annexin V staining, and cleaved caspase-3 immunoblotting using mixed lymphocyte reaction-stimulated peripheral blood lymphocytes (PBL) from healthy volunteers and lamina propria T cells from patients with Crohns disease. RESULTS Both infliximab and etanercept neutralized TNF-alpha effectively. Infliximab bound to activated PBL and lamina propria T cells, whereas binding of etanercept was equal to a nonspecific control antibody. Infliximab but not etanercept induced peripheral and lamina propria lymphocyte apoptosis when compared with a control antibody. Infliximab activated caspase 3 in a time-dependent manner, whereas etanercept did not. CONCLUSIONS Although both infliximab and etanercept showed powerful TNF-alpha neutralization, only infliximab was able to bind to PBL and lamina propria T cells and subsequently to induce apoptosis of activated lymphocytes. These data may provide a biological basis for the difference in efficacy of the 2 TNF-alpha-neutralizing drugs.

New Fundamentals in Hemostasis

Hemostasis encompasses the tightly regulated processes of blood clotting, platelet activation, and vascular repair. After wounding, the hemostatic system engages a plethora of vascular and extravascular receptors that act in concert with blood components to seal off the damage inflicted to the vasculature and the surrounding tissue. The first important component that contributes to hemostasis is the coagulation system, while the second important component starts with platelet activation, which not only contributes to the hemostatic plug, but also accelerates the coagulation system. Eventually, coagulation and platelet activation are switched off by blood-borne inhibitors and proteolytic feedback loops. This review summarizes new concepts of activation of proteases that regulate coagulation and anticoagulation, to give rise to transient thrombin generation and fibrin clot formation. It further speculates on the (patho)physiological roles of intra- and extravascular receptors that operate in response to these proteases. Furthermore, this review provides a new framework for understanding how signaling and adhesive interactions between endothelial cells, leukocytes, and platelets can regulate thrombus formation and modulate the coagulation process. Now that the key molecular players of coagulation and platelet activation have become clear, and their complex interactions with the vessel wall have been mapped out, we can also better speculate on the causes of thrombosis-related angiopathies.

Disulfide isomerization switches tissue factor from coagulation to cell signaling

Cell-surface tissue factor (TF) binds the serine protease factor VIIa to activate coagulation or, alternatively, to trigger signaling through the G protein-coupled, protease-activated receptor 2 (PAR2) relevant to inflammation and angiogenesis. Here we demonstrate that TF·VIIa-mediated coagulation and cell signaling involve distinct cellular pools of TF. The surface-accessible, extracellular Cys186–Cys209 disulfide bond of TF is critical for coagulation, and protein disulfide isomerase (PDI) disables coagulation by targeting this disulfide. A TF mutant (TF C209A) with an unpaired Cys186 retains TF·VIIa signaling activity, and it has reduced affinity for VIIa, a characteristic of signaling TF on cells with constitutive TF expression. We further show that PDI suppresses TF coagulant activity in a nitric oxide-dependent pathway, linking the regulation of TF thrombogenicity to oxidative stress in the vasculature. Furthermore, a unique monoclonal antibody recognizes only the noncoagulant, cryptic conformation of TF. This antibody inhibits formation of the TF·PAR2 complex and TF·VIIa signaling, but it does not prevent coagulation activation. These experiments delineate an upstream regulatory mechanism that controls TF function, and they provide initial evidence that TF·VIIa signaling can be specifically inhibited with minimal effects on coagulation.

Epidemiology of cancer-associated venous thrombosis

Cancer-associated venous thrombosis is a common condition, although the reported incidence varies widely between studies depending on patient population, start and duration of follow-up, and the method of detecting and reporting thrombotic events. Furthermore, as cancer is a heterogeneous disease, the risk of venous thrombosis depends on cancer types and stages, treatment measures, and patient-related factors. In general, cancer patients with venous thrombosis do not fare well and have an increased mortality compared with cancer patients without. This may be explained by the more aggressive type of malignancies associated with this condition. It is hypothesized that thromboprophylaxis in cancer patients might improve prognosis and quality of life by preventing thrombotic events. However, anticoagulant treatment leads to increased bleeding, particularly in this patient group, so in case of proven benefit of thromboprophylaxis, only patients with a high risk of venous thrombosis should be considered. This review describes the literature on incidence of and risk factors for cancer-associated venous thrombosis, with the aim to provide a basis for identification of high-risk patients and for further development and refinement of prediction models. Furthermore, knowledge on risk factors for cancer-related venous thrombosis may enhance the understanding of the pathophysiology of thrombosis in these patients.

The relationship between tissue factor and cancer progression: insights from bench and bedside

It is now widely recognized that a strong correlation exists between cancer and aberrant hemostasis. Patients with various types of cancers, including pancreatic, colorectal, and gastric cancer, often develop thrombosis, a phenomenon commonly referred to as Trousseau syndrome. Reciprocally, components from the coagulation cascade also influence cancer progression. The primary initiator of coagulation, the transmembrane receptor tissue factor (TF), has gained considerable attention as a determinant of tumor progression. On complex formation with its ligand, coagulation factor VIIa, TF influences protease-activated receptor-dependent tumor cell behavior, and regulates integrin function, which facilitate tumor angiogenesis both in vitro and in mouse models. Furthermore, evidence exists that an alternatively spliced isoform of TF also affects tumor growth and tumor angiogenesis. In patient material, TF expression and TF cytoplasmic domain phosphorylation correlate with disease outcome in many, but not in all, cancer subtypes, suggesting that TF-dependent signal transduction events are a potential target for therapeutic intervention in selected types of cancer. In this review, we summarize our current understanding of the role of TF in tumor growth and metastasis, and speculate on anticancer therapy by targeting TF.

Mechanistic View of Risk Factors for Venous Thromboembolism

Venous thromboembolism is an episodic disease with an annual incidence of 2 to 3/1000 per year that is associated with a high morbidity and mortality. Risk factors for venous thromboembolism come in many guises. They fit into an extended version of Virchows triad and they tilt the hemostatic balance toward clot formation. This can be achieved by decreasing blood flow and lowering oxygen tension, by activating the endothelium, by activating innate or acquired immune responses, by activating blood platelets, or by increasing the number of platelets and red blood cells or modifying the concentrations of pro- and anticoagulant proteins in the blood. In this narrative review we will discuss the known common risk factors within this pathophysiological framework.

Validity of bioluminescence measurements for noninvasive in vivo imaging of tumor load in small animals

A relatively new strategy to longitudinally monitor tumor load in intact animals and the effects of therapy is noninvasive bioluminescence imaging (BLI). The validity of BLIf or quantitative assessment of tumor load in small animals is critically evaluated in the present review. Cancer cells are grafted in mice or rats after transfection with a luciferase gene--usually that of a firefly. To determine tumor load, animals receive the substrate agent luciferin intraperitoneally, which luciferase converts into oxyluciferin in an ATP-dependent manner Light emitted by oxyluciferin in viable cancer cells is captured noninvasively with a highly sensitive charge-coupled device (CCD) camera. Validation studies indicate that BLI is useful to determine tumor load in the course of time, with each animal serving as its own reference. BLI is rapid, easy to perform, and sensitive. It can detect tumor load shortly after inoculation, even when relatively few cancer cells (2500-10,000) are used. BLI is less suited for the determination of absolute tumor mass in an animal because of quenching of bioluminescence by tissue components and the exact location of tumors because its spatial resolution is limited. Nevertheless, BLI is a powerful tool for high-throughput longitudinal monitoring of tumor load in small animals and allows the implementation of more advanced orthotopic tumor models in therapy intervention studies with almost the same simplicity as when measuring traditional ectopic subcutaneous models in combination with calipers.

Alternatively spliced tissue factor induces angiogenesis through integrin ligation

The initiator of coagulation, full-length tissue factor (flTF), in complex with factor VIIa, influences angiogenesis through PAR-2. Recently, an alternatively spliced variant of TF (asTF) was discovered, in which part of the TF extracellular domain, the transmembrane, and cytoplasmic domains are replaced by a unique C terminus. Subcutaneous tumors produced by asTF-secreting cells revealed increased angiogenesis, but it remained unclear if and how angiogenesis is regulated by asTF. Here, we show that asTF enhances angiogenesis in matrigel plugs in mice, whereas a soluble form of flTF only modestly enhances angiogenesis. asTF dose-dependently upregulates angiogenesis ex vivo independent of either PAR-2 or VIIa. Rather, asTF was found to ligate integrins, resulting in downstream signaling. asTF-αVβ3 integrin interaction induces endothelial cell migration, whereas asTF-dependent formation of capillaries in vitro is dependent on α6β1 integrin. Finally, asTF-dependent aortic sprouting is sensitive to β1 and β3 integrin blockade and a TF-antibody that disrupts asTF-integrin interaction. We conclude that asTF, unlike flTF, does not affect angiogenesis via PAR-dependent pathways but relies on integrin ligation. These findings indicate that asTF may serve as a target to prevent pathological angiogenesis.

Protease-Activated Receptor (PAR) 2, but not PAR1, Signaling Promotes the Development of Mammary Adenocarcinoma in Polyoma Middle T Mice

The G protein-coupled protease-activated receptors (PAR) are key signaling components for proteases in vascular biology and tumor progression. To address the contributions of PAR1 and PAR2 to breast cancer development, we established cohorts of mouse mammary tumor virus-polyoma middle T (PyMT) PAR1(-/-) and PAR2(-/-) mice, considering that the PyMT model recapitulates aspects of human disease. Appearance of palpable tumors, tumor expansion, and metastasis was indistinguishable between wild-type and PAR1(-/-) mice. PAR1(-/-) breast cancer cells were no longer responsive to thrombin in vitro, excluding compensatory up-regulation of alternative thrombin receptors and indicating that thrombin-PAR1 signaling is dispensable in breast tumor microenvironments. In contrast, palpable tumors and multifocal disease developed slower in PAR2(-/-) mice, and as a consequence of delayed tumor onset, metastasis was reduced. Analysis of early tumors showed persistence of adenomas with delayed appearance of vascularized adenocarcinomas in PAR2(-/-) mice. Furthermore, CXCL1 production by early PAR2(-/-) tumors was reduced. These results are consistent with previous xenograft data that implicated breast cancer PAR2 signaling in the induction of proangiogenic growth factors and chemokines. This study establishes that protease signaling contributes to mammary tumor development and that PAR2, rather than the thrombin receptor PAR1, plays a crucial role in the angiogenic switch.

Tissue Factor Coagulant Function Is Enhanced by Protein-disulfide Isomerase Independent of Oxidoreductase Activity *

Protein-disulfide isomerase (PDI) switches tissue factor (TF) from coagulation to signaling by targeting the allosteric Cys186–Cys209 disulfide. Here, we further characterize the interaction of purified PDI with TF. We find that PDI enhances factor VIIa-dependent substrate factor X activation 5–10-fold in the presence of wild-type, oxidized soluble TF but not TF mutants that contain an unpaired Cys186 or Cys209. PDI-accelerated factor Xa generation was blocked by bacitracin but not influenced by inhibition of vicinal thiols, reduction of PDI, changes in redox gradients, or covalent thiol modification of reduced PDI by N-ethylmaleimide or methyl-methanethiosulfonate, which abolished PDI oxidoreductase but not chaperone activity. PDI had no effect on fully active TF on either negatively charged phospholipids or in activating detergent, indicating that PDI selectively acts upon cryptic TF to facilitate ternary complex formation and macromolecular substrate turnover. PDI activation was reduced upon mutation of TF residues in proximity to the macromolecular substrate binding site, consistent with a primary interaction of PDI with TF. PDI enhanced TF coagulant activity on microvesicles shed from cells, suggesting that PDI plays a role as an activating chaperone for circulating cryptic TF.