Xanthones from the heartwood of Ochrocarpos odoratus

The heartwood of Ochrocarpos odoratus (Rafin) Merrill contains seven xanthones; 2-hydroxyxanthone, 3-hydroxy-2-methoxyxanthone, 1,5-dihydroxyxanthone, 2,3,4-trihydroxyxanthone, 1,5,6-trihydroxyxanthone, and 1,3,5,6- and 1,3,6,7-tetrahydroxyxanthones.     

Minor xanthones of Hypericum mysorense

2-Hydroxyxanthone, 1,7-dihydroxyxanthone, 1-hydroxy-7-methoxyxanthone, 6,7-dimethoxy-1-hydroxyxanthone and a new natural product, 2-hydroxy-3-methoxyxanthone, have been isolated and characterized from the phenolic fraction of the chloroform extract of the timber of Hypericum mysorense. The presence of simple xanthones in this genus supports the classification of Hypericum in the subfamily Hypericoideae in Guttiferae.     

Xanthones from Polygala alpestris (Rchb.)

Bioactivity-guided fractionation of Polygala alpestris L. (Rchb.) extracts led to the identification of two new xanthones, 1,3,7-trihydroxy-2,6-dimethoxyxanthone (1) and 2,3-methylenedioxy-4,7-dihydroxyxanthone (2). In addition five known compounds 3,4-dimethoxy-1,7-dihydroxyxanthone (3), 1,3-dihydroxy-7-methoxyxanthone (4), 1,7-dihydroxy-2,3-dimethoxyxanthone (5), 3',6-O-disinapoyl sucrose (6) and 3',5'-dimethoxybiphenyl-4-olo (7) were isolated. The structures of the isolated compounds were established by means of high resolution mass spectrometry, mono- and bi-dimensional NMR spectroscopy. All isolated compounds were tested for cytotoxic activity against three tumor cell lines (LoVo, HL-60, K 562).     

Biologically active alkylated coumarins from Kayea assamica

Four coumarin derivatives, theraphins A (1), B (2), C (3), and D (4), along with three known xanthones, 2-hydroxyxanthone, 1,7-dihydroxyxanthone, and 5-hydroxy-1-methoxyxanthone, were isolated from the bark of Kayea assamica (Clusiaceae) native to Myanmar. Their structures were determined using spectroscopic and chemical techniques. The absolute configuration of 1 was established by the modified Mosher ester method. Theraphins A (1), B (2), and C (3) exhibited good cytotoxicity against Col2, KB, and LNCaP human cancer cell lines. Theraphin D (4) showed mild activity only against the KB cell line. The coumarins also exhibited mild antimalarial activities.     

Thrombin, TNF-alpha, and LPS exert overlapping but nonidentical effects on gene expression in endothelial cells and vascular smooth muscle cells

Thrombin, TNF-alpha, and LPS have each been implicated in endothelial cell and vascular smooth muscle cell (VSMC) activation. We wanted to test the hypothesis that these three agonists display mediator and/or cell type-specific properties. The addition of thrombin to human pulmonary artery endothelial cells resulted in an upregulation of PDGF-A, tissue factor (TF), ICAM-1, and urokinase-type plasminogen activator (u-PA), whereas TNF-alpha and LPS failed to induce PDGF-A. These effects were mimicked by protease-activated receptor-1 activation. In VSMC, thrombin induced expression of TF and PDGF-A but failed to consistently induce ICAM-1 or u-PA expression. In contrast, TNF-alpha and LPS increased expression of all four genes in this cell type. Inhibitor studies in endothelial cells demonstrated a critical role for PKC in mediating thrombin, TNF-alpha, and LPS induction of ICAM-1, TF, and u-PA and for p38 MAPK in mediating thrombin, TNF-alpha, and LPS induction of TF. Taken together, these results suggest that inflammatory mediators engage distinct signaling pathways and expression profiles in endothelial cells and VSMC. The data support the notion that endothelial cell activation is not an all-or-nothing phenomenon but rather is dependent on the nature of the extracellular mediator.     

Cerebral endothelial cells release TNF-alpha after stimulation with cell walls of Streptococcus pneumoniae and regulate inducible nitric oxide synthase and ICAM-1 expression via autocrine loops.

TNF-alpha, inducible NO synthase (iNOS), and ICAM-1 are considered to be key proteins in the inflammatory response of most tissues. We tested the hypothesis that cell walls of Streptococcus pneumoniae (PCW), the most common cause of adult bacterial meningitis, induce TNF-alpha, iNOS, and ICAM-1 expression in rat primary brain microvascular endothelial cell cultures. We detected TNF-alpha mRNA by RT-PCR already 1 h after stimulation with PCW, while TNF-alpha protein peaked at 4 h (9.4 +/- 3.6 vs 0.1 +/- 0.1 pg/microgram protein). PCW induced iNOS mRNA 2 h after stimulation, followed by an increase of the NO degradation product nitrite (18.1 +/- 4 vs 5.8 +/- 1.8 at 12 h; 18.1 +/- 4 vs 5.8 +/- 1.8 pmol/microgram protein at 72 h). The addition of TNF-alpha Ab significantly reduced nitrite production to 62.2 +/- 14.4% compared with PCW-stimulated brain microvascular endothelial cells (100%). PCW induced the expression of ICAM-1 (measured by FACS), which was completely blocked by TNF-alpha Ab (142 +/- 18.6 vs 97.5 +/- 12.4%; 100% unstimulated brain microvascular endothelial cells). Cerebral endothelial cells express TNF-alpha mRNA as well as iNOS mRNA and release the bioactive proteins in response to PCW. PCW-induced NO production is mediated in part by an autocrine pathway involving TNF-alpha, whereas ICAM-1 expression is completely mediated by this autocrine loop. By these mechanisms, cerebral endothelial cells may regulate critical steps in inflammatory blood-brain-barrier disruption of bacterial meningitis.     

Effect of high glucose concentrations on expression of ELAM-1, VCAM-1 and ICAM-1 in HUVEC with and without cytokine activation

Diabetes mellitus is associated with an increased prevalence of endothelial dysfunction and development of atherosclerotic vascular diseases. We demonstrate here that hyperglycemia results in the expression of adhesion molecules on endothelial cells in vitro. Incubation of human umbilical vein endothelial cells (HUVEC) in a culture medium with 11.0 mM, 16.5 mM and 22.0 mM glucose concentrations induced the expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and endothelial-leukocyte adhesion molecule-1 (ELAM-1). This effect was detectable after 24 h incubation of HUVEC with a high glucose concentration. The effect of high glucose concentration on TNF-alpha induced expression of ELAM-1, VCAM-1 and ICAM-1 was negligible, if at all. These results show that even a short-term exposure of endothelial cells (ECs) to high glucose concentration leads to their activation associated with increased expression of adhesion molecules such as ELAM-1, VCAM-1 and ICAM-1.     

Effects of TNF-alpha on leukocyte adhesion molecule expressions in cultured human lymphatic endothelium.

TNF-alpha alters leukocyte adhesion molecule expression of cultured endothelial cells like human umbilical vein endothelial cells (HUVEC). This study was designed to investigate the changes in vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and platelet endothelial cell adhesion molecule-1 (PECAM-1) expression with TNF-alpha stimulation in cultured human neonatal dermal lymphatic endothelial cells (HNDLEC). The real-time quantitative PCR analysis on HNDLEC showed that TNF-alpha treatment leads to increases of VCAM-1 and ICAM-1 mRNAs to the 10.8- and 48.2-fold levels of untreated cells and leads to a reduction of PECAM-1 mRNA to the 0.42-fold level of untreated cells. Western blot and immunohistochemical analysis showed that TNF-alpha leads to VCAM-1 and ICAM-1 expressions that were inhibited by antiserum to human TNF receptor or by AP-1 inhibitor nobiletin. In flow cytometry analysis, the number of VCAM-1- and ICAM-1-positive cells increased, and PECAM-1-positive cells decreased with TNF-alpha treatment. Regarding protein amounts produced in cells and amounts expressed on the cell surface, VCAM-1 and ICAM-1 increased in HNDLEC and HUVEC, and PECAM-1 decreased in HNDLEC in a TNF-alpha concentration-dependent manner. VCAM-1, ICAM-1, and PECAM-1 protein amounts in TNF-alpha-stimulated cells were lower in HNDLEC than in HUVEC. This suggests that the lymphatic endothelium has the TNF-alpha-induced signaling pathway, resulting in increased VCAM-1 and ICAM-1 expression to a weaker extent than blood endothelium and PECAM-1 reduction to a stronger extent than blood endothelium.     

Haemoglobin-induced oxidative stress is associated with both endogenous peroxidase activity and H2O2 generation from polyunsaturated fatty acids

Patients with increased haemolytic haemoglobin (Hb) have 10-20-times greater incidence of cardiovascular mortality. The objective of this study was to evaluate the role of Hb peroxidase activity in LDL oxidation. The role of Hb in lipid peroxidation, H(2)O(2) generation and intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) was assessed using NaN(3), a peroxidase inhibitor, catalase, a H(2)O(2) decomposing enzyme and human umbilical vein endothelial cells (HUVECs), respectively. Hb induced H(2)O(2) production by reacting with LDL, linoleate and cell membrane lipid extracts. Hb-induced LDL oxidation was inhibited by NaN(3) and catalase. Furthermore, Hb stimulated ICAM-1 and VCAM-1 expression, which was inhibited by the antioxidant, probucol. Thus, the present study suggests that the peroxidase activity of Hb produces atherogenic, oxidized LDL and oxidized polyunsaturated fatty acids (PUFAs) in the cell membrane and reactive oxygen species (ROS) formation mediated Hb-induced ICAM-1 and VCAM-1 expression.     

Effects of antioxidants and nitric oxide on TNF-alpha-induced adhesion molecule expression and NF-kappaB activation in human dermal microvascular endothelial cells

Cell adhesion molecules expressed on endothelial cells in inflamed skin appear to be controlled by the actions of cytokines and reactive oxygen species. However, molecular mechanisms of the expression of adhesion molecules during skin inflammation are currently not well understood. To evaluate the role of antioxidants and nitric oxide in modulating inflammatory processes in the skin, we examined the effects of pyrrolidine dithiocarbamate (PDTC, 0.1 mM) and spermine NONOate (Sper-NO, 1 mM) on adhesion molecule expression and nuclear factor kappa B (NF-kappaB) activation induced by TNF-alpha (10 ng/ml) in cultured human dermal microvascular endothelial cells (HDMEC). Treatment of cells with TNF-alpha for 4 h significantly induced the surface expression of E-selectin and intercellular adhesion molecule-1 (ICAM-1). Treatment with TNF-alpha for 8 h significantly induced the surface expression of E-selectin, ICAM-1 and vascular cell adhesion molecule-1 (VCAM-1). The up-regulation of these adhesion molecules was suppressed significantly by pretreatment with PDTC or Sper-NO for 1 h. The mRNA expression of E-selectin, ICAM-1 and VCAM-1, and activation of NF-kappaB induced by TNF-alpha for 2 h were significantly decreased by the above two pretreatments. N-acetylcysteine (10 mM) and S-nitroso-N-acetylpenicillamine (1 mM) had no significant inhibitory effects on the cell surface and mRNA expression of these adhesion molecules stimulated by TNF-alpha. These findings indicate that both cell surface and mRNA expression of adhesion molecules in HDMEC induced by TNF-alpha are inhibited significantly by pretreatment with PDTC or Sper-NO, possibly in part through blocking the activation of NF-kappaB. These results suggest a potential therapeutic approach using antioxidant agents or nitric oxide pathway modulators in the treatment of inflammatory skin diseases.     

Novel thiocoumarins as inhibitors of TNF-alpha induced ICAM-1 expression on human umbilical vein endothelial cells (HUVECs) and microsomal lipid peroxidation

Different coumarin/thiocoumarin derivatives, that is, 7-hydroxy-4-methylcoumarin, 7,8-dihydroxy-4-methylcoumarin, 7-acetoxy-4-methylcoumarin, 7,8-diacetoxy-4-methylcoumarin, 7-hydroxy-4-methylthiocoumarin, 7,8-dihydroxy-4-methylthiocoumarin, 7-acetoxy-4-methylthiocoumarin and 7,8-diacetoxy-4-methylthiocoumarin were synthesized and evaluated for their effects on TNF-alpha induced expression of intercellular adhesion molecule-1 (ICAM-1) on endothelial cells and on NADPH-catalyzed rat liver microsomal lipid peroxidation with a view to identify modulators for expression of cell adhesion molecules and to establish structure-activity relationship. We found that dihydroxy and diacetoxy derivatives of thiocoumarin were more potent in comparison to the corresponding coumarin derivatives in inhibiting TNF-alpha-induced expression of ICAM-1. However, coumarin derivatives were found to be more potent in comparison to the corresponding thiocoumarins in inhibiting microsomal lipid peroxidation. We have also tested the intermediate compounds 7,8-dibenzyloxy-4-methylcoumarin and 7,8-dibenzyloxy-4-methylthiocoumarin for their inhibitory activity on TNF-alpha-induced ICMA-1 expression. We found that dibenzyloxy-4-methylthiocoumarin is better than dibenzyloxy-4-methylcoumarin. The mechanisms underlying the observed activities of coumarins and thiocoumarins have been discussed with reference to their structures. Such structure-function relationship studies may help in developing molecules with better anti-inflammatory and anti-oxidant activities.     

Respiratory syncytial virus infection of human lung endothelial cells enhances selectively intercellular adhesion molecule-1 expression

Respiratory syncytial virus (RSV) is worldwide the most frequent cause of bronchiolitis and pneumonia in infants requiring hospitalization. In the present study, we supply evidence that human lung microvascular endothelial cells, human pulmonary lung aorta endothelial cells, and HUVEC are target cells for productive RSV infection. All three RSV-infected endothelial cell types showed an enhanced cell surface expression of ICAM-1 (CD54), which increased in a time- and RSV-dose-dependent manner. By using noninfectious RSV particles we verified that replication of RSV is a prerequisite for the increase of ICAM-1 cell surface expression. The up-regulated ICAM-1 expression pattern correlated with an increased cellular ICAM-1 mRNA amount. In contrast to ICAM-1, a de novo expression of VCAM-1 (CD106) was only observed on RSV-infected HUVEC. Neither P-selectin (CD62P) nor E-selectin (CD62E) was up-regulated by RSV on human endothelial cells. Additional experiments performed with neutralizing Abs specific for IL-1alpha, IL-1beta, IL-6, and TNF-alpha, respectively, excluded an autocrine mechanism responsible for the observed ICAM-1 up-regulation. The virus-induced ICAM-1 up-regulation was dependent on protein kinase C and A, PI3K, and p38 MAPK activity. Adhesion experiments using polymorphonuclear neutrophil granulocytes (PMN) verified an increased ICAM-1-dependent adhesion rate of PMN cocultured with RSV-infected endothelial cells. Furthermore, the increased adhesiveness resulted in an enhanced transmigration rate of PMN. Our in vitro data suggest that human lung endothelial cells are target cells for RSV infection and that ICAM-1 up-regulated on RSV-infected endothelial cells might contribute to the enhanced accumulation of PMN into the bronchoalveolar space.     

TNF-alpha and IL-1alpha induce apoptosis in subconfluent rat mesangial cells. Evidence for the involvement of hydrogen peroxide and lipid peroxidation as second messengers

Apoptosis of mesangial cells (MC) plays a role in glomerulonephritis (GN). In this study we investigated cytokine-induced apoptosis of cultured rat MC by morphological and biochemical features. TNF-alpha and IL-1alpha induced apoptosis in rat MC in a time- and concentration-dependent fashion. RT-PCR experiments revealed that MC express the TNF-receptor 1 (p60) gene constitutively. TNF-alpha as well as IL-1alpha stimulated the production of reactive oxygen species (ROS) and induced lipid peroxidation. Coincubation with catalase inhibited TNF-alpha and IL-1alpha induced apoptosis as well as lipid peroxidation. TNF-alpha, but not IL-1alpha increased the expression of c-jun. These results provide evidence that TNF-alpha and IL-1alpha induce apoptosis in rat MC with hydrogen peroxide and lipid peroxidation as second messengers. Increased c-jun expression may be a downstream intracellular signal of TNF-alpha-, but not IL-1alpha-induced apoptosis.     

Mast cells induce upregulation of P-selectin and intercellular adhesion molecule 1 on carotid endothelial cells in a new in vitro model of mast cell to endothelial cell communication

It is suggested that mast cells contribute to cell recruitment in inflammation through the upregulation of endothelial adhesion molecules. P-selectin and intercellular adhesion molecule(ICAM)-1 are two key adhesion molecules that have been associated indirectly with mast cell activity. The canine C2 mastocytoma cell line and primary cultures of canine carotid endothelial cells were used to establish a new in vitro model to help study the interaction between mast cells and endothelial cells. Carotid endothelial cells were incubated with mast cell mediators to uncover their effect on endothelial ICAM-1 and P-selectin expression. To assess the relative contributions of tumour necrosis factor (TNF)-alpha and histamine to such effect, an H1 antihistamine and a TNF-alpha blocking antibody were used. Prior to activation by mast cell mediators, P-selectin was expressed only within the cytoplasm, and ICAM-1 was constitutively expressed on the surface of the canine carotid endothelial cells. Both adhesion molecules were enhanced significantly and strongly upon mast cell activation at various time points. Unstored TNF-alpha was fully responsible for ICAM-1 upregulation. P-selectin was up-regulated by both preformed and newly synthesized mast cell mediators, but neither histamine nor TNF-alpha accounted for such an effect. Therefore,a new model is proposed in which the pro-inflammatory effect of mast cells on endothelial cells can be studied in vitro.In this model, it has been demonstrated that only TNF-alpha accounts for the overexpression of ICAM-1 induced by mast cells, and that mast cells up-regulate P-selectin expression through a histamine-independent mechanism.     

De novo ICAM-1 synthesis in the mouse lung: model of assessment of protein expression in lungs

Because most studies addressing the regulatory mechanisms of intercellular adhesion molecule (ICAM)-1 expression have used cultured endothelial cells, we set out to develop an isolated mouse lung preparation to study gene and protein expression in its proper cellular context in the organ. Lungs from CD1 mice were isolated and perfused (2 ml/min, 37 degrees C) with a recirculating volume of RPMI 1640 solution supplemented with 3 g/100 ml albumin. Lungs maintained their isogravimetric state for 4 h. Tumor necrosis factor (TNF-alpha; 2,000 U/ml) was added to the perfusate for 0.5, 1, 2, or 3.5 h to induce ICAM-1 expression or lungs received no treatment (control). After quick-freezing the lungs using liquid nitrogen at different time points, the prepared tissue homogenates were analyzed for ICAM-1 protein expression by Western blotting and NF-kappaB activation by electrophoretic mobility shift assay. TNF-alpha caused a progressive increase in NF-kappaB activity after 0.5 h and ICAM-1 protein expression two- to threefold of basal after 2 h. Untreated lungs expressed a low and constant level of ICAM-1 between 0 and 3.5 h. TNF-alpha failed to induce NF-kappaB activation and ICAM-1 expression in lungs of NADPH oxidase-deficient mice lacking p47(phox). We disaggregated mouse lungs using collagenase and stained the cells for ICAM-1 and VE-cadherin (used as an endothelial marker) to assess the in situ endothelial-specific expression of ICAM-1. We observed that TNF-alpha challenge resulted in increased ICAM-1 expression in endothelial cells freshly isolated from lungs. These data show the role of NADPH oxidase-derived oxidant signaling in the mechanism of NF-kappaB activation and ICAM-1 expression in mouse lung endothelial cells. Moreover, the general method presented herein has potential value in assessing mechanisms of gene and protein expression in the isolated-perfused mouse lung model.