The effects of COX-metabolites on cyclooxygenase-2 induction in LPS-treated endothelial cells.

Cyclooxygenase (COX) is the first enzyme in the pathway in which arachidonic acid is converted to PGs, also called COX-metabolites. COX exists as COX-1 and COX-2 isoforms. Each COX-metabolite has different characters and functions. The amounts of each COX-metabolite produced in cells are also different depending on cell type and mitogen stimulated cells. These were thought to be autoregulation among COX-metabolites. Here, we have investigated the effects of COX-metabolites, such as PGI2, PGE2, PGF2alpha and U44069, on the induction of COX-2 in human umbilical vein endothelial cells (HUVEC) treated with LPS (1 microg/ml). COX activity was measured by the production of 6-keto-PGF1alpha, PGE2, PGF2alpha and TXB2 in the presence of exogenous arachidonic acids (10 microM for 10 min) using enzyme immunoassay (EIA). COX-1 and COX-2 protein was measured by immunoblotting using specific antibody. PGI2, PGE2, PGF2alpha or U44069, did not affect on basal COX activity in untreated HUVEC (24 h incubation). Untreated HUVEC contained COX-1 protein but not COX-2 protein. When HUVEC were treated with LPS (1 microg/ml for 24 h), COX activity and COX-2 protein was increased in a dose dependent manner. The increased COX activity in LPS (1 microg/ml) treated HUVEC was inhibited with PGE2 (0.03, 0.3 or 3 microM), but not PGI2, PGF2alpha or U44069, in a dose dependent manner. Similarly, COX-2 protein expression in LPS treated HUVEC was also inhibited with PGE2, but not PG2, PGF2alpha or U44069, in a dose dependent manner. These results suggested that PGE2, but not PGI2, PGF2alpha or TXA2 is a key in feedback regulation of COX-metabolites produced in HUVEC.

The activation of platelet aggregation by human cholangiocarcinoma cells is mediated through thrombin receptor.

Tumor cell induced platelet aggregation (TCIPA) played an importance role in early state of thrombosis in cancer patients. In addition, TCIPA was recognized as one important step in metastatic cascade. Cholangiocarcinoma, one of the most common cancers in the north-eastern part of Thailand, associated with thrombosis was reported. The authors investigated the effects of cholangiocarcinoma cells on platelet function as measured by platelet aggregation. Primary human cholangiocarcinoma (HuCCA) cells were established in our laboratory. Cells were cultured as standard techniques and grown to confluence until used, after which cells were replaced with fresh medium (Dulbeco Modified Eargle's Medium, DMEM) without serum for 24, 48 and 72 h. Then, the conditioned medium (CM) was collected. CM (24, 48 and 72 h) from HuCCA failed to induce platelet aggregation, whereas, HuCCA pellets induced platelet aggregation and potentiated platelet aggregation induced by submaximal concentration of thrombin. Interestingly, platelet aggregation induced by HuCCA was inhibited by hirudin (thrombin receptor antagonist; 10, 20 and 40 U) in a dose dependent manner. Thus, cholangiocarcinoma cells can induce platelet aggregation in a direct tumor cell-platelet contact via thrombin receptor. Therefore, the use of antiplatelet agents especially via thrombin receptors may help to prevent TCIPA or metastasis by CCA.

Serum nitric oxide levels in patients with coronary artery disease.

Nitric oxide (NO) plays a pivotal role in the pathophysiology of coronary artery disease. The roles of NO are not only physiological but also pathological in the cardiovascular system. An inappropriate release of NO has been linked to the pathogenesis of CAD. The authors investigated whether serum NOx (nitrate and nitrite), a stable end product of NO, level was related to patients with coronary artery disease. The blood chemistry, such as cholesterol, triglyceride, LDL-C, HDL-C and blood sugar, was also measured in comparison with serum NOx. Serum NOx was measured in samples from 20 healthy controls, 20 angina patients without angiographic evidence of coronary lesions (CAG) and 20 angina patients with angiographic evidence of coronary lesions (CAD) by using modified Griess reaction. The mean serum NOx levels in the CAD groups was higher than CAG and control groups (41.3 +/- 5.5, 32.7 +/- 3.9 and 25.7 +/- 3.5 micromol/L, respectively). NOx levels in the CAD group was only significantly higher than the control groups (p < 0.05) but not the CAG groups. There were no significant differences of NOx levels in all age groups. In the CAD group, women showed significantly higher NOx levels than men (64.0 +/- 7.5 and 29.0 +/- 4.7 microl/L, respectively, p < 0.05). Interestingly, the mean serum NOx levels in the CAD groups was significantly higher in a group of abnormal lipid profiles (cholesterol, triglyceride, LDL-C) and blood sugar than in a group of normal profiles. The results suggested that there was an increased NOx levels in patients with coronary artery disease and much higher in patients with multiple underlying conditions such as hyperlipidemia and hyperglycemia. Thus, the measurement of the NOx levels at different times may help to monitor the state and severity of coronary artery disease.

Effects of Russell's viper venom on mediator production in cultured human umbilical vein endothelial cells.

Hemodynamic alterations in Russell's viper envenomation are the result of interactions of various vasoactive mediators and perhaps proinflammatory cytokines. Since vascular endothelium is likely to be exposed to high concentrations of the venom and the endothelial cell itself not only plays an important role in the physiologic control of the circulation, but also play a role in inflammation with the synthesis and secretion of proinflammatory cytokines. It was therefore, the objective of this study to determine the effects of Russell's viper venom (RVV) on proinflammatory cytokine production by cultured human umbilical vein endothelial cells (HUVEC) and the release of endothelium-derived substances. Endothelial cells were isolated from freshly obtained human umbilical cord vein and grown in tissue culture to confluence as a homogeneous population. Cells were then incubated at 37 degrees C under 5 per cent CO2 with RVV (0.2, 1.0, 5.0, and 25.0 microg/ml) or lipopolysaccharide (LPS, 10 microg/ml) for 3, 6, 12 and 24 hours. After an indicated time, the levels of endothelin-1 (ET-1); 6-keto-PGF1alpha (a stable metabolite of PGI2) tumor necrosis factor-alpha (TNF-alpha); interleukin-1beta (IL-1beta); and interleukin-6 (IL-6) in supernatants were measured by using ELISA or EIA. The effect of RVV or LPS on cell viability was also measured using MIT assay. The results showed copious amounts of ET-1 production irrespectively with the presence of RVV or LPS. Whereas, production of PGI2 (measured as 6-keto-PGF1alpha, a stable metabolite) was increased significantly higher in the RVV- and LPS-treated EC than in the control EC. However, TNF-alpha and IL-6 productions were not different among these groups. The levels of IL-1beta were very low, although IL-1beta was detectable in the group treated with RVV at a concentration of 25.0 microg/ml. In conclusion, RVV upto 25 microg/ml stimulated PGI2 production by cultured HUVEC. This effect was unlikely related to production of proinflammatory cytokines since LPS or RVV is not sufficient per se to elevate a substantial amount of EC-derived cytokines. The higher amount of IL-6 compared to TNF-alpha and IL-1beta may be produced through other pathways apart from production via a cascade of cytokines. This is the first report showing that RVV up to 25 microg/ml has no effect on prominent proinflammatory cytokine production by HUVEC. However, in blood circulation, the major source of cytokines production is monocyte-macrophage lineage cell. Thus, RVV in blood circulation may activate the production of proinflammatory cytokines mainly from those cells and subsequently induce toxicity.

The induction of cyclooxygenase-2 (COX-2) in cultured endothelial cells treated with serum from preeclampsia is mediated by interleukin-6.

COX-2 protein, but not COX-1 protein, was induced in HUVEC from women with a normal pregnancy (nHUVEC) treated with serum from patients with preeclampsia (pSerum), but not with serum from women with a normal pregnancy (nSerum). COX activity in pSerum treated nHUVEC was less than in nSerum treated nHUVEC. Interestingly, the induction of COX-2 protein in nHUVEC treated with pSerum was inhibited by antiIL-6 antibody. The decreased COX activity in nHUVEC treated with pSerum plus antiIL-6 antibody was also reversed in a dose dependent manner. Thus, the induction of COX-2 in pSerum treated nHUVEC was mediated by IL-6. Therefore, the development of selective inhibitors of COX-2 or of IL-6 antagonists may have a potential role in the prevention and treatment of preeclampsia.

The expression of cyclooxygenase-2 in human umbilical vein endothelial cell culture from preeclampsia.

We have shown the HUVEC from normal pregnancy contained COX-1 protein but not COX-2 protein and released 6-keto-PGF1 alpha 277 +/- 5 ng/ml (for 24 h). In contrast, HUVEC from preeclampsia contained both COX-1 and COX-2 protein and released significantly lesser amounts of 6-keto-PGF1 alpha (159 +/- 8 ng/ml for 24 h; p < 0.05). Thus, COX-2 is expressed in HUVEC from preeclampsia but not in normal pregnancy and affects the release of prostacyclin suggesting the involvement of COX-2 in the pathogenesis of preeclampsia. The development of selective inhibitors of COX-2 may have a potential role in prevention and treatment of preeclampsia.