L. Vijaya Mohan Rao

Localization of human tissue factor antigen by immunostaining with monospecific, polyclonal anti-human tissue factor antibody

Tissue factor, the cofactor for factor VIIa-catalyzed activation of factors IX and X, plays an important role in the initiation of hemostasis. However, the distribution of tissue factor in the body has not been defined until recently. In the present study frozen sections of non-malignant human tissues were immunostained using polyclonal, monospecific rabbit anti-human tissue factor antibodies. Specificity of the anti-tissue factor antibody was established by Western blotting. Sensitivity of the immunostaining technique for tissue factor antigen was confirmed by correlating staining of non-perturbed and perturbed cultured human umbilical vein endothelial cells with their surface membrane tissue factor coagulant activity. Brain, lung and placenta, all known to possess large amounts of tissue factor procoagulant activity, stained strongly for tissue factor, as did peripheral nerves and autonomic ganglia. Epithelium of skin, mucosa, and glomeruli also stained; however, epithelium lining excretory ducts failed to stain. Skeletal muscle did not stain, but cardiac muscle stained faintly. Smooth muscle also did not stain except for the muscularis mucosa of the esophagus, which stained brightly. Fibroblasts varied in stainability; those found in the adventitia of vessels stained strongly. The endothelium, tunica intima and tunica media of blood vessels consistently failed to stain. The distribution of tissue factor antigen as demonstrated by immunostaining supports the hypothesis that maintenance of a physical barrier between tissue factor activity and blood is key to the normal regulation of hemostasis.

Resveratrol, a Polyphenolic Compound Found in Wine, Inhibits Tissue Factor Expression in Vascular Cells A Possible Mechanism for the Cardiovascular Benefits Associated With Moderate Consumption of Wine

A number of studies suggest that moderate consumption of red wine may be more effective than other alcoholic beverages in decreasing the risk of coronary heart disease mortality. The phytochemical resveratrol found in wine, derived from grapes, has been thought to be responsible for cardiovascular benefits associated with wine consumption because it was shown to have antioxidant and antiplatelet activities. In the present investigation, we examined the effect of resveratrol on induction of tissue factor (TF) expression in vascular cells that were exposed to pathophysiological stimuli. The data presented herein show that resveratrol, in a dose-dependent manner, inhibited the expression of TF in endothelial cells stimulated with a variety of agonists, including interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNFalpha) and lipopolysaccharide (LPS). A similar inhibition of TF induction was also seen in LPS stimulated monocytes that were pretreated with resveratrol before their stimulation with LPS. In addition, resveratrol was shown to inhibit the LPS-induced expression of TNFalpha mRNA in endothelial cells and of TNFalpha and IL-1beta mRNA in monocytes. Nuclear run-on analysis in endothelial cells showed that resveratrol inhibited TF expression at the level of transcription. However, resveratrol did not significantly alter the binding of the transcription factors c-Fos/c-Jun and c-Rel/p65, the transcription factors required for the induction of TF promoter in both endothelial cells and monocytes. Similarly, resveratrol had no significant effect on the binding of NF-kappaB in endothelial cells stimulated with IL-1beta, TNFalpha, and LPS. Overall, our data show that resveratrol could effectively suppress the aberrant expression of TF and cytokines in vascular cells, but it requires further investigation to understand how resveratrol exerts its inhibitory effect.

Tissue Factor–Factor VIIa Signaling

How does tissue factor (TF), whose principle role is to support clotting factor VIIa (FVIIa) in triggering the coagulation cascade, affect various pathophysiological processes? One of the answers is that TF interaction with FVIIa not only initiates clotting but also induces cell signaling via activation of G-protein-coupled protease activated receptors (PARs). Recent studies using various cell model systems and limited in vivo systems are beginning to define how TF-VIIa-induced signaling regulates cellular behavior. Signaling pathways initiated by both TF-VIIa protease activation of PARs and phosphorylation of the TF-cytoplasmic domain appear to regulate cellular functions. In the present article, we review the emerging data on the mechanism of TF-mediated cell signaling and how it regulates various cellular responses, with particular focus on TF-VIIa protease-dependent signaling.

Endothelial Cell Protein C Receptor Acts as a Cellular Receptor for Factor VIIa on Endothelium

Although factor VII/factor VIIa (FVII/FVIIa) is known to interact with many non-vascular cells, activated monocytes, and endothelial cells via its binding to tissue factor (TF), the interaction of FVII/FVIIa with unperturbed endothelium and the role of this interaction in clearing FVII/FVIIa from the circulation are unknown. To investigate this, in the present study we examined the binding of radiolabeled FVIIa to endothelial cells and its subsequent internalization. 125I-FVIIa bound to non-stimulated human umbilical vein endothelial cells (HUVEC) in time- and dose-dependent manner. The binding is specific and independent of TF and negatively charged phospholipids. Protein C and monoclonal antibodies to endothelial cell protein C receptor (EPCR) blocked effectively 125I-FVIIa binding to HUVEC. FVIIa binding to EPCR is confirmed by demonstrating a marked increase in 125I-FVIIa binding to CHO cells that had been stably transfected with EPCR compared with the wild-type. Binding analysis revealed that FVII, FVIIa, protein C, and activated protein C (APC) bound to EPCR with similar affinity. FVIIa binding to EPCR failed to accelerate FVIIa activation of factor X or protease-activated receptors. FVIIa binding to EPCR was shown to facilitate FVIIa endocytosis. Pharmacological concentrations of FVIIa were found to impair partly the EPCR-dependent protein C activation and APC-mediated cell signaling. Overall, the present data provide convincing evidence that EPCR serves as a cellular binding site for FVII/FVIIa. Further studies are needed to evaluate the pathophysiological consequences and relevance of FVIIa binding to EPCR.

IL-22 Produced by Human NK Cells Inhibits Growth of Mycobacterium tuberculosis by Enhancing Phagolysosomal Fusion

We determined whether human NK cells could contribute to immune defenses against Mycobacterium tuberculosis through production of IL-22. CD3−CD56+ NK cells produced IL-22 when exposed to autologous monocytes and γ-irradiated M. tuberculosis, and this depended on the presence of IL-15 and IL-23, but not IL-12 or IL-18. IL-15-stimulated NK cells expressed 10.6 times more DAP10 mRNA compared with control NK cells, and DAP10 siRNA inhibited IL-15-mediated IL-22 production by NK cells. Soluble factors produced by IL-15-activated NK cells inhibited growth of M. tuberculosis in macrophages, and this effect was reversed by anti-IL-22. Addition of rIL-22 to infected macrophages enhanced phagolysosomal fusion and reduced growth of M. tuberculosis. We conclude that NK cells can contribute to immune defenses against M. tuberculosis through production of IL-22, which inhibits intracellular mycobacterial growth by enhancing phagolysosomal fusion. IL-15 and DAP-10 elicit IL-22 production by NK cells in response to M. tuberculosis.

Inhibition of Tissue Factor Gene Activation in Cultured Endothelial Cells by Curcumin Suppression of Activation of Transcription Factors Egr-1, AP-1, and NF-κB

Binding of plasma factor VII(a) to tissue factor (TF) initiates the coagulation cascade. In health, TF is not expressed in endothelial cells. However, endothelial cells express TF in response to lipopolysaccharide (LPS), tumor necrosis factor-alpha (TNF alpha), and other biological stimuli. TF expression by endothelial cells is implicated in thrombotic disorders in patients with a variety of clinical disorders. In the present study, we demonstrate that curcumin (diferulolylmethane), a known anticarcinogenic and anti-inflammatory agent, inhibited phorbol 12-myristate 13-acetate (PMA), LPS, TNF alpha, and thrombin-induced TF activity and TF gene transcription in human endothelial cells. The present data show that curcumin prevented the activation of c-Rel/p65, which is essential for TF gene activation in endothelial cells, by impairing the proteolytic degradation inhibitor protein, I kappa B alpha. The data also show that curcumin downregulated AP-1 binding activity. The present studies are the first to demonstrate that PMA, but not LPS, TNF alpha, and thrombin, induced Egr-1 binding to the second serum-responsive region (SRR-2) of TF promoter and that curcumin inhibited the PMA-induced Egr-1 binding to SRR-2. Overall, the data suggest that the anticarcinogenic and anti-inflammatory properties of curcumin may be related to its ability to inhibit cellular gene expression regulated by transcription factors NF-kappa B, AP-1, and Egr-1.

Binding of annexin V to a human ovarian carcinoma cell line (OC-2008). Contrasting effects on cell surface factor VIIa/tissue factor activity and prothrombinase activity

Proteins of the annexin/lipocortin family bind tightly to anionic phospholipids and platelets and act as in vitro anticoagulants. Annexins may be useful as tools to study the availability of anionic phospholipids on cell surfaces and their role in the regulation of blood coagulation. In the present study, we investigated the binding of annexin V (placental anticoagulant protein I) to a human ovarian carcinoma cell line, OC-2008, that constitutively expresses surface membrane tissue factor activity. Binding of annexin V to cell monolayers was calcium-dependent, specific, saturable and reversible; Scatchard analysis indicated a single class of binding sites with an apparent Kd of 9.4 +/- 3.1 nM and 5.2 +/- 1 x 10(6) sites per cell. Binding was completely inhibited by phospholipid vesicles containing phosphatidylserine, but was not inhibited by vesicles containing phosphatidylcholine. Annexin V inhibited the cell surface-dependent activity of prothrombinase complex, but did not inhibit the activity of the factor VIIa/tissue factor complex. In conclusion, these results suggest that anionic phospholipid is present on the extracellular face of OC-2008 cells; this anionic phospholipid is functionally important for the activity of the prothrombinase complex, but the importance of anionic phospholipid for the cell surface factor VIIa/tissue factor functional activity is unclear.

Plasmin Induces Cyr61 Gene Expression in Fibroblasts Via Protease-Activated Receptor-1 and p44/42 Mitogen-Activated Protein Kinase–Dependent Signaling Pathway

Objective—The plasminogen system has been proposed to participate in vascular remodeling and angiogenesis. Although plasmin-mediated proteolysis could contribute these processes, proteolytic targets for plasmin and their downstream effector molecules are yet to be fully defined. The aim of the present study was to elucidate potential mechanisms by which plasmin affects various cellular processes. Methods and Results—Plasmin upregulated the expression of Cyr61, a growth factor–like gene that has been implicated in cell proliferation, adhesion, and migration. Plasmin-induced gene expression is dependent on its proteolytic activity and requires its binding to cells. Studies that used wild-type fibroblasts and fibroblasts derived from PAR-1– and PAR-2–deficient mice showed that plasmin induced Cyr61 gene expression in wild-type fibroblasts and PAR-2–deficient cells but not in PAR-1–deficient cells. Consistent with this, plasmin induced the activation of p44/42 mitogen-activated protein kinase in wild-type, PAR-2 −/− cells but not in PAR-1 −/− cells. In contrast with thrombin, plasmin failed to induce Ca2+ signaling in fibroblasts. Conclusions—Plasmin induced an angiogenic and wound-healing promoter, Cyr61, in fibroblasts through activation of PAR-1. Plasmin-induced Cyr61 expression is mediated via the p44/42 mitogen-activated protein kinase pathway independent of Ca2+ signaling.

Suppression of transcription factor Egr-1 by curcumin.

The transcription factor early growth response-1 gene product (Egr-1) is a member of the family of immediate early response genes and regulates a number of pathophysiologically relevant genes in vasculature that are involved in growth, differentiation, immune response, wound healing, and blood clotting. In the present study, we investigated the effect of curcumin, a natural plant phenolic compound known to exhibit anticarcinogenic, antioxidant, and antiinflammatory properties, on Egr-1 expression in endothelial cells and fibroblasts. Gel mobility shift assays showed that pretreatment of endothelial cells and fibroblasts with curcumin suppressed phorbol 12-myristate 13-acetate and serum-induced Egr-1 binding activity to the consensus Egr-1 binding site and also to the Egr-1 binding site present in the promoter of tissue factor gene. Western blot analysis revealed that curcumin inhibited phorbol 12-myristate 13-acetate-induced de novo synthesis of Egr-1 protein in endothelial cells. Suppression of Egr-1 protein expression in curcumin-treated cells stemmed from the suppression of Egr-1 mRNA. Northern blot analysis showed that curcumin inhibited serum and phorbol 12-myristate 13-acetate induced expression of tissue factor and urokinase-type plasminogen activator receptor mRNA in fibroblasts. Cumulatively, the data show that curcumin suppresses the induction of transcription factor Egr-1 and thereby modulates the expression of Egr-1-regulated genes in endothelial cells and fibroblasts.

Endothelial cell protein C receptor: a multiliganded and multifunctional receptor

Endothelial cell protein C receptor (EPCR) was first identified and isolated as a cellular receptor for protein C on endothelial cells. EPCR plays a crucial role in the protein C anticoagulant pathway by promoting protein C activation. In the last decade, EPCR has received wide attention after it was discovered to play a key role in mediating activated protein C (APC)-induced cytoprotective effects, including antiapoptotic, anti-inflammatory, and barrier stabilization. APC elicits cytoprotective signaling through activation of protease activated receptor-1 (PAR1). Understanding how EPCR-APC induces cytoprotective effects through activation of PAR1, whose activation by thrombin is known to induce a proinflammatory response, has become a major research focus in the field. Recent studies also discovered additional ligands for EPCR, which include factor VIIa, Plasmodium falciparum erythrocyte membrane protein, and a specific variant of the T-cell receptor. These observations open unsuspected new roles for EPCR in hemostasis, malaria pathogenesis, innate immunity, and cancer. Future research on these new discoveries will undoubtedly expand our understanding of the role of EPCR in normal physiology and disease, as well as provide novel insights into mechanisms for EPCR multifunctionality. Comprehensive understanding of EPCR may lead to development of novel therapeutic modalities in treating hemophilia, inflammation, cerebral malaria, and cancer.