Aryl Hydrocarbon Receptor Activation and Tissue Factor Induction by Fluid Shear Stress and Indoxyl Sulfate in Endothelial Cells.

Endogenous agonists of the transcription factor aryl hydrocarbon receptor (AHR) such as the indolic uremic toxin, indoxyl sulfate (IS), accumulate in patients with chronic kidney disease. AHR activation by indolic toxins has prothrombotic effects on the endothelium, especially via tissue factor (TF) induction. In contrast, physiological AHR activation by laminar shear stress (SS) is atheroprotective. We studied the activation of AHR and the regulation of TF by IS in cultured human umbilical vein endothelial cells subjected to laminar fluid SS (5 dynes/cm2). SS and IS markedly increased the expression of AHR target genes PTGS2 (encoding for COX2), AHRR, CYP1A1, and CYP1B1, as well as F3 (encoding for TF), in an AHR-dependent way. IS amplified SS-induced TF mRNA and protein expression and upregulation of AHR target genes. Interestingly, tyrosine kinase inhibition by genistein decreased SS- but not IS-induced TF expression. Finally, the increase in TF expression induced by laminar SS was not associated with increased TF activity. In contrast, IS increased TF activity, even under antithrombotic SS conditions. In conclusion, IS and SS induce AHR activation and AHR-dependent TF upregulation by different mechanisms. Impairment of the antithrombotic properties of shear stressed endothelium by toxic AHR agonists could favor cardiovascular diseases in CKD.

Protective effect of Myrtle (Myrtus communis) on burn induced skin injury.

Thermal skin burns cause local injury as well as triggers acute systemic inflammation response where the imbalance between oxidative and antioxidative system occurs. As an alternative treatment, various medicinal herbs are used to treat burn injuries in many countries. In this study, the possible protective role of oral or topical Myrtle (Myrtus communis L.) treatment against burn-induced damage was investigated. The dorsum of the Wistar Albino rats was shaved and exposed to 90 °C water bath in burn group or 25 °C water bath in control group for 10 s under ether anesthesia. Myrtle extract was applied 100 mg/kg/day for 2 days either orally or topically. In skin samples; malondialdehyde and glutathione levels, catalase, superoxide dismutase, nitric oxide and tissue factor activities were determined. Skin tissues were also examined by light microscopy. Severe thermal skin burn injury caused a significant decrease in glutathione level, superoxide dismutase, catalase and tissue factor activities as well as nitric oxide level, which was accompanied with significant increases in skin malondialdehyde level. Myrtle treatment reversed all these biochemical indices except topical Myrtle treated group's nitric oxide level, as well as histopathological alterations, which were induced by thermal trauma. Both oral and topical Myrtle extract treatment was found to have protective role in the burn induced oxidative injury, which may be attributed to the potential antioxidant effect of Myrtle. As a conclusion, Myrtle significantly diminishes burn-induced damage in skin.

α-1 Antitrypsin Enhances Islet Engraftment by Suppression of Instant Blood-Mediated Inflammatory Reaction.

Islet cell transplantation has limited effectiveness because of an instant blood-mediated inflammatory reaction (IBMIR) that occurs immediately after cell infusion and leads to dramatic ß-cell death. In intraportal islet transplantation models using mouse and human islets, we demonstrated that α-1 antitrypsin (AAT; Prolastin-C), a serine protease inhibitor used for the treatment of AAT deficiency, inhibits IBMIR and cytokine-induced inflammation in islets. In mice, more diabetic recipients reached normoglycemia after intraportal islet transplantation when they were treated with AAT compared with mice treated with saline. AAT suppressed blood-mediated coagulation pathways by diminishing tissue factor production, reducing plasma thrombin-antithrombin complex levels and fibrinogen deposition on islet grafts, which correlated with less graft damage and apoptosis. AAT-treated mice showed reduced serum tumor necrosis factor-α levels, decreased lymphocytic infiltration, and decreased nuclear factor (NF)-κB activation compared with controls. The potent anti-inflammatory effect of AAT is possibly mediated by suppression of c-Jun N-terminal kinase (JNK) phosphorylation. Blocking JNK activation failed to further reduce cytokine-induced apoptosis in ß-cells. Taken together, AAT significantly improves islet graft survival after intraportal islet transplantation by mitigation of coagulation in IBMIR and suppression of cytokine-induced JNK and NF-κB activation. AAT-based therapy has the potential to improve graft survival in human islet transplantation and other cellular therapies on the horizon.

The Antithrombotic Potential of Tinzaparin and Enoxaparin Upon Thrombin Generation Triggered In Vitro by Human Ovarian Cancer Cells IGROV1.

BACKGROUND: A documented relationship between ovarian cancer and thrombosis does exist. Low-molecular-weight heparins (LMWHs) are cornerstone drugs in the primary prevention and treatment of venous thromboembolic events in patients with cancer. However, cancer cells may alter the efficiency of these antithrombotic agents. OBJECTIVE: We aimed to characterize the procoagulant phenotype of human epithelial ovarian adenocarcinoma cells, IGROV1, and to compare the capacity of tinzaparin and enoxaparin to inhibit thrombin generation triggered by these cells. METHODS: Thrombin generation induced by different concentrations of IGROV1 cells on platelet poor plasma (PPP) was assessed by the calibrated automated thrombogram assay. Tissue factor (TF) expression was studied using Western blot analysis. Then, the experimental model of thrombin generation was used to compare the inhibitory effect of clinically relevant concentrations of both tinzaparin and enoxaparin. The inhibitory concentration 50 (IC50) of the mean rate index and the endogenous thrombin potential and the 2-fold increase in lag time were analyzed on the basis of the anti-Xa and anti-IIa activities of the LMWHs. RESULTS: IGROV1 cells suspended into PPP resulted in a significant increase in thrombin generation in the absence of any exogenous source of TF and phospholipids. Tissue factor was expressed by IGROV1 cells. Tinzaparin was a more potent inhibitor of thrombin generation than enoxaparin. The inhibition of thrombin generation induced by IGROV1 cancer cells depended mainly on the anti-Xa activity of the LMWHs. CONCLUSION: This experimental study in ovarian cancer cells demonstrates that the antithrombotic activity of LMWHs is not completely predicted by the anti-Xa or anti-IIa activities measured in PPP.

Tissue factor expression in obese type 2 diabetic subjects and its regulation by antidiabetic agents.

OBJECTIVE: Increased coagulation activation may contribute to the high incidence of cardiovascular complications observed in obese and type 2 diabetes (T2D) subjects. Although tissue factor (TF), the primary initiator of coagulation is increased in obesity, its expression in adipose tissues and its association with metabolic parameters are unclear. We sought to compare TF expression in plasma and adipose tissues of obese subjects with and without T2D, its correlation with metabolic parameters, and regulation in response to antidiabetic drugs. METHODS: Subjects were recruited from diabetes clinics and adipose tissue was obtained by needle biopsy of lower subcutaneous abdominal depot. For the intervention study, subjects were randomized into treatment groups with rosiglitazone or metformin for 4 months. RESULTS: Plasma TF antigen, activity, and adipose TF mRNA were greater in obese T2D subjects compared with obese nondiabetics. Plasma TF activity correlated with fasting insulin, glucose, and free fatty acids, (FFAs), and adipose TF mRNA correlated with plasma FFA. Plasma TF activity was reduced by metformin and increased with rosiglitazone treatment. CONCLUSIONS: Specific diabetes-related metabolic parameters, but not obesity per se, are correlated with TF expression. Regulation of TF activity by different classes of antidiabetic drugs may relate to protective or adverse cardiovascular outcomes.

Effective treatment of chemoresistant breast cancer in vitro and in vivo by a factor VII-targeted photodynamic therapy.

BACKGROUND: The purpose of this study was to test a novel, dual tumour vascular endothelial cell (VEC)- and tumour cell-targeting factor VII-targeted Sn(IV) chlorin e6 photodynamic therapy (fVII-tPDT) by targeting a receptor tissue factor (TF) as an alternative treatment for chemoresistant breast cancer using a multidrug resistant (MDR) breast cancer line MCF-7/MDR. METHODS: The TF expression by the MCF-7/MDR breast cancer cells and tumour VECs in MCF-7/MDR tumours from mice was determined separately by flow cytometry and immunohistochemistry using anti-human or anti-murine TF antibodies. The efficacy of fVII-tPDT was tested in vitro and in vivo and was compared with non-targeted PDT for treatment of chemoresistant breast cancer. The in vitro efficacy was determined by a non-clonogenic assay using crystal violet staining for monolayers, and apoptosis and necrosis were assayed to elucidate the underlying mechanisms. The in vivo efficacy of fVII-tPDT was determined in a nude mouse model of subcutaneous MCF-7/MDR tumour xenograft by measuring tumour volume. RESULTS: To our knowledge, this is the first presentation showing that TF was expressed on tumour VECs in chemoresistant breast tumours from mice. The in vitro efficacy of fVII-tPDT was 12-fold stronger than that of ntPDT for MCF-7/MDR cancer cells, and the mechanism of action involved induction of apoptosis and necrosis. Moreover, fVII-tPDT was effective and safe for the treatment of chemoresistant breast tumours in the nude mouse model. CONCLUSIONS: We conclude that fVII-tPDT is effective and safe for the treatment of chemoresistant breast cancer, presumably by simultaneously targeting both the tumour neovasculature and chemoresistant cancer cells. Thus, this dual-targeting fVII-tPDT could also have therapeutic potential for the treatment of other chemoresistant cancers.

Challenges and approaches for assay development of membrane and membrane-associated proteins in drug discovery.

In addition to its role as a barrier between the cytoplasm and the extracellular milieu, the cell membrane is a scaffold for a diverse collection of receptors and enzymes. The organization afforded by this scaffold serves to ensure an efficient interaction between the components of the membrane. The desire to maintain this organization in solution is a challenge for the appropriate interrogation of these biochemical components. This chapter will discuss strategies that allow biochemical analysis of membrane-associated enzymes within standard biochemical reactions. The advantages of these screening strategies in identifying valuable compounds from compound libraries and in understanding the intricacies of complex multiprotein complexes (i.e., chemotaxis) will be discussed.

Weight-adjusted dalteparin for prevention of vascular thromboembolism in advanced pancreatic cancer patients decreases serum tissue factor and serum-mediated induction of cancer cell invasion.

The aim of the present study was to assess the role of tissue factor and serum-induced cell invasion in patients with advanced pancreatic cancer (APC). A cohort of 39 patients with APC, without thrombosis, receiving chemotherapy, were entered in a randomized controlled trial (ISRCTN = 76464767) of thromboprevention with weight-adjusted dalteparin (WAD). A total of 19 patients received WAD, the remaining 20 acting as a control group. Serum from baseline and week 8 was analysed for circulating-tissue factor antigen using ELISA. Circulating-tissue factor antigen rose from 324 pg/ml, [interquartile range (IQR) 282-347 pg/ml] to 356 pg/ml, (IQR 319-431 pg/ml) in controls (C), and decreased in the dalteparin-treated group (D) from 336 pg/ml (IQR 281-346 pg/ml) to 303 pg/ml (IQR 274-339 pg/ml). The difference in median percentage change between D and C was statistically significant [-4.0 (D) vs. 4.7 (C); P = 0.005, n = 39]. Serum-induced cellular invasion of MIA-Paca-2 cells in response to patient serum was studied using a Boyden chamber assay in 30 patients (14 WAD and 16 C). The median percentage change between C and D was significant [+54.9 (C) vs. -21.9 (D) P = 0.025, n = 30]. There was a weak correlation between BB-tissue factor reduction and cellular invasion reduction (Spearman) [0.384 (P = 0.037, n = 30)]. APC patients treated with WAD have lower tissue factor antigen levels and attenuated induction of cellular invasion in their blood. These assays may provide useful markers to guide appropriate dalteparin (and other low-molecular weight heparin) dosing schedules to optimize anticancer effects of dalteparin in APC.

Paclitaxel potentiates inflammatory cytokine-induced prothrombotic molecules in endothelial cells.

To overcome the limitations of balloon expandible metal stent-induced neointimal smooth muscle cell proliferation, drug-coated stent devices have been developed. Drug eluting stents release high concentrations of antiproliferative agents, such as paclitaxel, to reduce neointimal hyperplasia. The proinflammatory cytokine, tumor necrosis factor-alpha (TNF-alpha), is known to cause severe endothelial dysfunction and accelerate atherosclerotic lesion progression. The interaction of TNF-alpha and paclitaxel on the release of prothrombotic molecules was examined in endothelial cells. Treatment of endothelial cells with paclitaxel had no direct effect on tissue factor (TF) expression, but TNF-alpha increased TF. Cotreatment of paclitaxel with TNF-alpha markedly augmented the release of TF. TNF-alpha induced release of plasminogen activator inhibitor but no synergism occurred with paclitaxel. Treatment of endothelial cells with paclitaxel and TNF-alpha reduced expression of thrombomodulin and protein C receptor. Tissue factor pathway inhibitor expression was reduced by prolonged treatment with either paclitaxel or TNF-alpha. The adhesion molecule, CD62 E, was induced by TNF-alpha; however, CD31, CD62 P, and CD106 were not affected by paclitaxel and TNF-alpha. Apoptosis was not observed with cotreatment of endothelial cells with paclitaxel and TNF-alpha. CD59-positive microparticles were released in response to TNF-alpha, but the release was not augmented by paclitaxel. Paclitaxel and TNF-alpha increased the nitrotyrosination of proteins. These findings indicate that paclitaxel enhances TNF-alpha-induced release of TF, and downregulated thrombomodulin, increased protein nitration, which may subsequently favor prothrombotic intimal surface.

[Effect of tanshinone II A on NB4 cell induced procoagulant activity in human umbilical vein endothelial cells].

OBJECTIVE: To investigate the effect of tanshinone II A on the procoagulant activity (PCA) of human umbilical vein endothelial cells (HUVEC) induced by acute promyelocytic leukemia (APL) cell line NB4 cells. METHODS: The HUVEC were incubated for 6, 12, and 24 hours in different tanshinone II A conditioned medias (Tan II A-NB4-24h-CM, Tan II A-NB4-72h-CM, Tan II A-NB4-120h-CM). Then the HUVEC were incubated for 6, 12, 24, and 72 hours with Tan II A-NB4-120h-CM and different concentrations of Tan II A (0, 0.25, 0.5, 1.0 microg/mL). The HUVEC lysates were obtained by three repeated freezing and thrawing. Their PCA were tested using the one stage clotting assay. The activity of tissue factor (TF : act) was tested using the chromogenic substrate assay. The control groups included 0.3 microg/mL ATRA, 0.01% DMSO and RPMI 1640. RESULTS: Tan II A-(72 h,120 h)-NB4-CM elevated PCA of HUVEC and six hours of incubation in the 120 h-NB4-CM had the greatest PCA. The PCA of HUVEC in the 1.0 microg/mL Tan II A-NB4-CM was the same as in the 0.3 microg/mL ATRA-NB4-CM. (2) The NB4-CM induced PCA of HUVEC decreased with 5.0 microg/mL of Tan II A, at a level similar to the decrease with 0.3 microg/mL of ATRA. Less than 5.0 microg/mL of Tan II did not reduce the NB4-CM induced PCA of HUVEC. (3) Both Tan II A 120 h-NB4-CM and ATRA 120 h-NB4-CM elevated the TF : act of HUVEC. The TF : act reached the peak after 6 hours of incubation. The Tan II A 120 h-NB4-CM maintained the peak level of TF : act at the 12th hour and fell to the base line at the 24th hour. The ATRA 120 h-NB4-CM induced TF:act dropped down with time after reaching its peak at the 6th hour. (4) The 1.0 microg/mL of Tan II A did not reduce the TF : act of HUVEC induced by the Tan II A 120 h-NB4-CM. But the 0.3 microg/mL of ATRA reduced the TF : act of HUVEC at the 6th hour. CONCLUSION: TanIIA-NB4-CM increases PCA and TF : Act of HUVEC. TanIIA decreases PCA of HUVECs induces by TanIIA-NB4-CM.

Paclitaxel downregulates tissue factor in cancer and host tumour-associated cells.

Paclitaxel, a microtubule-stabilising compound with potent anti-tumour activity, has been clinically used in a wide variety of malignancies. Tissue factor (TF) is often expressed by tumour-associated endothelial and inflammatory cells, as well as by cancer cells themselves, and it is considered a hallmark of cancer progression. We investigated whether paclitaxel could modulate TF in human mononuclear (MN) cells, human umbilical vein endothelial cells (HUVEC) and the metastatic breast carcinoma cell line MDA-MB-231. Cells were incubated with or without paclitaxel at 37 degrees C. At the end of incubation, cells were disrupted and tested for procoagulant activity by a one-stage clotting assay, for TF antigen levels by ELISA and TF mRNA by real-time RT-PCR. IL-6 and IL-1beta were tested by ELISA in conditioned medium. Both the strong TF activity and antigen constitutively expressed by MDA-MB-231 and the TF induced by LPS, TNF-alpha and IL-1beta in MN cells and HUVEC were significantly reduced by paclitaxel. In the presence of paclitaxel, lower TF mRNA levels were also detected. Since paclitaxel has been shown to induce the expression of inflammatory genes in monocytes and tumour cells, we tested whether paclitaxel could influence IL-6 and IL-1beta release from the cells used in this paper. Neither the constitutive expression of IL-6 and IL-1beta by MDA-MB-231 nor the basal and LPS-induced release from MN cells and HUVEC was affected. Our data support the hypothesis that the anti-tumour effects of paclitaxel may, at least in part, be mediated by the capacity of this drug to modulate the procoagulant potential of cancer and host cells.

Guggulsterone, an anti-inflammatory phytosterol, inhibits tissue factor and arterial thrombosis.

BACKGROUND: The phytosterol guggulsterone is a potent anti-inflammatory mediator with less side effects than classic steroids. This study assesses the impact of guggulsterone on tissue factor (TF) expression and thrombus formation. METHODS AND RESULTS: Guggulsterone inhibited TNF-alpha-induced endothelial TF protein expression and surface activity in a concentration-dependent manner; in contrast, dexamethasone did not affect TNF-alpha-induced TF expression. Guggulsterone enhanced endothelial tissue factor pathway inhibitor and impaired plasminogen activator inhibitor-1 as well as vascular cell adhesion molecule-1 protein. Real-time polymerase chain reaction revealed that guggulsterone inhibited TNF-alpha-induced TF mRNA expression; moreover, it impaired activation of the MAP kinases JNK and p38, while that of ERK remained unaffected. In vivo, guggulsterone inhibited TF activity and photochemical injury induced thrombotic occlusion of mouse carotid artery. Guggulsterone also inhibited TF expression, proliferation, and migration of vascular smooth muscle cells in a concentration-dependent manner. CONCLUSIONS: Guggulsterone inhibits TF expression in vascular cells as well as thrombus formation in vivo; moreover, it impairs vascular smooth muscle cell activation. Hence, this phytosterol offers novel therapeutic options, in particular in inflammatory diseases associated with an increased risk of thrombosis.

Tissue factor and PAR signaling in tumor progression.

Tumor development depends on multiple reciprocal interactions of tumor cells with the host cell compartment. Tumor cells initiate TF-dependent crosstalks with the tumor microenvironment by releasing procoagulant microparticles, soluble cytokines and angiogenic growth factors. Conversely, the hemostatic system in the host compartment provides multiple circuits that regulate tumor growth and sustain angiogenesis. A combination of experimental models of spontaneous and transplanted tumor development and metastasis start to delineate the role of TF in tumor progression and identified potential therapeutic approaches to target the TF pathway.

Effects of peroxisome proliferator-activated receptor ligands in modulating tissue factor and tissue factor pathway inhibitor in acutely symptomatic carotid atheromas.

BACKGROUND AND PURPOSE: Severely stenotic, symptomatic carotid atheromas are associated with a high risk of stroke in the short term. Although carotid endarterectomy is effective in reducing this stroke risk, it is frequently not applied within the time window for significant benefit. We investigated the effect of peroxisome proliferator-activated receptor (PPAR) -alpha and -gamma ligands in acutely modifying tissue factor (TF) and tissue factor pathway inhibitor (TFPI) in unstable carotid atheromas. METHODS: During a 3-year period, 64 patients who had experienced a transient ischemic attack or stroke with good recovery within 6 weeks before surgery and 12 asymptomatic patients with a >70% carotid stenosis were recruited. The expression of PPAR-alpha and -gamma was investigated in endarterectomy samples. The effects of the PPAR-alpha and -gamma ligands fenofibrate and rosiglitazone were investigated in cell culture experiments. Targeted biopsy specimens from endarterectomy samples (n=48) were incubated with medication for 4 days. TF and TFPI were assessed by immunohistochemistry, Western blot analysis, flow cytometry, and activity assays. RESULTS: PPAR-gamma1 but not -alpha was downregulated in atheromas removed from patients with recent symptoms and no evidence of diabetes. Fenofibrate but not rosiglitazone impaired the induction of TF in human endothelial cells and reduced resting levels of TF activity in vascular smooth muscle cells. Rosiglitazone but not fenofibrate increased TFPI secretion from human endothelial cells. Both fenofibrate (100+/-18.7% to 56.6+/-8.8%, P=0.005; 0.2664+/-0.0696 to 0.1771+/-0.0310, P=0.02) and rosiglitazone (100+/-22% to 88.3+/-20%, P=0.02; 0.3113+/-0.0729 to 0.2287+/-0.0415, P=0.04) reduced TF expression and activity, respectively, in atheroma biopsy specimens. A low expression of TFPI was found in atheroma biopsy specimens with little evidence of TFPI activity. CONCLUSIONS: This study suggests that both PPAR-alpha and -gamma ligands have beneficial effects in acutely reducing TF in unstable carotid atheromas.

Plasma level of stromal derived factor-1 (SDF-1) is increased in disseminated intravascular coagulation patients who have poor outcomes: in vitro effect of SDF-1 on coagulopathy.

Stromal cell-derived factor-1 (SDF-1) is a CXC chemokine that activates and directs the migration of leukocytes that have CXCR4, which is the unique receptor for SDF-1. Although SDF-1/CXCR4 interaction has been implicated in various inflammatory conditions, its role in modulating coagulation has not been determined. We studied the plasma SDF-1 levels in 90 patients with suspected disseminated intravascular coagulation (DIC) and we found that circulating SDF-1 was significantly increased in the overt DIC patients and was also increased in overt DIC patients who have a poor outcome. We then tested in vitro whether SDF-1 can affect the expression of monocyte tissue factor (TF) and endothelial thrombomodulin (TM), and both of these play important roles in coagulopathy. SDF-1 did not affect the expression of surface TF protein and its function and the TF mRNA level in both monocytes and the monocytic leukemia cell line THP-1. SDF-1 also did not change the surface TM expression of endothelial cells. SDF-1 could enhance low-dose ADP induced platelet aggregation, although it failed by itself to induce aggregation. These findings suggest that plasma SDF-1 might be closely associated with hypercoagulability though its action as a platelet activator.

N-Acetylcysteine derivative inhibits procoagulant activity of human islet cells.

AIMS/HYPOTHESIS: The early loss of beta cells after islet cell transplantation has been attributed in part to blood coagulation at the implant site. Tissue factor expressed by beta cells and contaminating duct cells is considered to activate this process. Here, we investigated the ability of N-acetyl-L-cysteine to suppress the in vitro procoagulant activity of duct cells and human islet cell preparations. MATERIALS AND METHODS: The effects of Nacystelyn, a salt derivative of N-acetyl-L-cysteine, were first assessed on procoagulant activity induced in human plasma by recombinant tissue factor, human primary duct cells or human islet cell preparations. The influence of Nacystelyn on clot formation, platelet counts and D-dimers were measured in a whole blood tubing loop model. Human beta cell viability and insulin synthesis after Nacystelyn treatment were assessed to exclude cytotoxicity of Nacystelyn. RESULTS: Nacystelyn efficiently inhibited the procoagulant activity of human recombinant tissue factor, primary duct cells and human islet cell preparations at clinically relevant concentrations without cellular toxicity. CONCLUSIONS/INTERPRETATION: Nacystelyn is a pharmaceutical candidate to reduce early beta cell loss related to tissue factor-dependent coagulation after islet transplantation.

[Platelet and tissue factor: review].

It is generally accepted that tissue factor plays an important role in coagulation and intravascular thrombus formation. Tissue factor is not only found primarily on the surface of certain cells that are located outside the vasculature, but also found in circulating cells. Monocyte express tissue factor induced by endotoxin. Recently, many researches indicate that P-selectin, CD40 ligand and GPIIb/IIIa receptor of platelet can also affect expression of tissue factor by monocyters. In addition, a lot of studies showed that tissue factor exist in the circulation including contained platelet. Tissue factor in the platelet releases under certain condition, and initiates coagulation. In this review the relation between platelet and tissue factor was elaborated.

The inhibitory effect of triterpenoid glycosides originating from Sanguisorba officinalis on tissue factor activity and the production of TNF-alpha.

We examined the inhibitory effects of novel triterpene glycoside compounds [ziyu-glycoside II (ZY-II) and its methyl ester (ZYM-201)], which originated from the roots of sanguisorba officinalis L. (Rosaceae), on tissue factor (TF) activity and tumor necrosis factor (TNF)-alpha production. In in vitro TF activity tests, ZY-II but not ZYM-201 strongly blocked lung TF activity with an IC50 value of 0.46 microM. By contrast, only ZYM-201 dose-dependently inhibited in vivo TF activity with an ED50 value of 1.7 mg/kg, when orally administered. Furthermore, ZYM-201 diminished both in vitro and in vivo TNF-alpha production with IC50 or ED50 values of 69.4 microM and 87.4 mg/kg, respectively. Therefore, these results suggest either that ZYM-201 may be developed as a potent inhibitor of both TF- and TNF-alpha-mediated diseases such as atherosclerosis and septic shock, or it may be a lead compound to be derivatized for further improvement of its curative efficacy.

Potentiation of angiogenic switch in capillary endothelial cells by cAMP: A cross-talk between up-regulated LLO biosynthesis and the HSP-70 expression.

During tumor growth and invasion, the endothelial cells from a relatively quiescent endothelium start proliferating. The exact mechanism of switching to a new angiogenic phenotype is currently unknown. We have examined the role of intracellular cAMP in this process. When a non-transformed capillary endothelial cell line was treated with 2 mM 8Br-cAMP, cell proliferation was enhanced by approximately 70%. Cellular morphology indicated enhanced mitosis after 32-40 h with almost one-half of the cell population in the S phase. Bcl-2 expression and caspase-3, -8, and -9 activity remained unaffected. A significant increase in the Glc(3)Man(9)GlcNAc(2)-PP-Dol biosynthesis and turnover, Factor VIIIC N-glycosylation, and cell surface expression of N-glycans was observed in cells treated with 8Br-cAMP. Dol-P-Man synthase activity in the endoplasmic reticulum membranes also increased. A 1.4-1.6-fold increase in HSP-70 and HSP-90 expression was also observed in 8Br-cAMP treated cells. On the other hand, the expression of GRP-78/Bip was 2.3-fold higher compared to that of GRP-94 in control cells, but after 8Br-cAMP treatment for 32 h, it was reduced by 3-fold. GRP-78/Bip expression in untreated cells was 1.2-1.5-fold higher when compared with HSP-70 and HSP-90, whereas that of the GRP-94 was 1.5-1.8-fold lower. After 8Br-cAMP treatment, GRP-78/Bip expression was reduced 4.5-4.8-fold, but the GRP-94 was reduced by 1.5-1.6-fold only. Upon comparison, a 2.9-fold down-regulation of GRP-78/Bip was observed compared to GRP-94. We, therefore, conclude that a high level of Glc(3)Man(9)GlcNAc(2)-PP-Dol, resulting from 8Br-cAMP stimulation up-regulated HSP-70 expression and down-regulated that of the GRP-78/Bip, maintained adequate protein folding, and reduced endoplasmic reticulum stress. As a result capillary endothelial cell proliferation was induced.