Athiwat Thaworn

The induction of cyclooxygenase-2 by 17β-estradiol in endothelial cells is mediated through protein kinase C

Abstract:Objective and Design: We investigated whether estrogen affected COX isoform expressed in human umbilical vein endothelial cells (HUVEC).¶Materials and Methods: HUVEC were grown to confluence and replaced with fresh medium containing 17β-estradiol (0.001, 0.01, 0.1 and 1 nM) or 17β-estradiol (1 nM) plus staurosporine (0.1, 1 and 10 ng/ml) for 24 h, after which the supernatant medium was collected to measure 6-keto-PGF1α using enzyme immunoassay. To measure COX activity via exogenous substrates, the remaining cells were replaced with fresh medium containing arachidonic acid (10 μM for 10 min), and then the medium was removed to measure 6-keto-PGF1α. The COX isoform expressed in cells was detected by immunoblotting using specific antibody.¶Results: 17β-estradiol (0.001 to 1 nM) increased the production of 6-keto-PGF1α via either endogenous or exogenous substrate in a dose dependent manner. These increases were significantly inhibited when cells were coincubated with staurosporine. Interestingly, only COX-2 protein, but not COX-1 protein, was induced in 17β-estradiol treated HUVEC and was also inhibited by staurosporine.¶Conclusion: Our data showed that 17β-estradiol increased the release of PGI2 from HUVEC via the induction of COX-2 which was mediated through protein kinase C. The results suggested that COX-2 might have a role in the cardiovascular protective effect of estrogen.¶

The induction of cyclooxygenase-2 in IL-1beta-treated endothelial cells is inhibited by prostaglandin E2 through cAMP.

Prostaglandins (PGs) have numerous cardiovascular and inflammatory effects. Cyclooxygenase (COX), which exists as COX-1 and COX-2 isoforms, is the first enzyme in the pathway in which arachidonic acid is converted to PGs. Prostaglandin E2 (PGE2) exerts a variety of biological activities for the maintenance of local homeostasis in the body. Elucidation of PGE2 involvement in the signalling molecules such as COX could lead to potential therapeutic interventions. Here, we have investigated the effects of PGE2 on the induction of COX-2 in human umbilical vein endothelial cells (HUVEC) treated with interleukin-1beta (IL-1beta 1 ng/ml). COX activity was measured by the production of 6-keto-PGF1alpha, PGE2, PGF2alpha and thromboxane B2 (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. Untreated HUVEC contained only COX-1 protein while IL-1beta treated HUVEC contained COX-1 and COX-2 protein. PGE2 (3 microM for 24h) did not affect on COX activity and protein in untreated HUVEC. Interestingly, PGE2 (3 microM for 24h) can inhibit COX-2 protein, but not COX-1 protein, expressed in HUVEC treated with IL-1beta. This inhibition was reversed by coincubation with forskolin (100 microM). The increased COX activity in HUVEC treated with IL-1beta was also inhibited by PGE2 (0.03, 0.3 and 3 microM for 24h) in a dose-dependent manner. Similarly, forskolin (10, 50 or 100 microM) can also reverse the inhibition of PGE2 on increased COX activity in IL-1beta treated HUVEC. The results suggested that (i) PGE2 can initiate negative feedback regulation in the induction of COX-2 elicited by IL-1beta in endothelial cells, (ii) the inhibition of PGE2 on COX-2 protein and activity in IL-1beta treated HUVEC is mediated by cAMP and (iii) the therapeutic use of PGE2 in the condition which COX-2 has been involved may have different roles.

The effects of estrone, estradiol and estriol on platelet aggregation induced by adrenaline and adenosine diphosphate

The impact of estrogens on the cardiovascular system and their ability to regulate platelet functions remains controversial. Changes in platelet functions could contribute to thrombotic risk associated with estrogen treatments. Here, we investigated the effects of various forms of estrogen, including estrone (E1), estradiol (E2) and estriol (E3), on platelet aggregation induced by standard agonists (adrenaline and adenosine diphosphate). Platelet-rich plasma (PRP) was prepared from citrated blood donated by 25 normal volunteers. The study on platelet aggregation was carried out in 96-well flat-bottom microtitre plates and assessed using a microplate reader. For studying the effects of each estrogen, PRP was preincubated with 1, 10 and 100 nM of E1, E2 and E3 at 37°C for 20 min, and then coincubated with normal saline (control untreated PRP), adrenaline (ADR) or adenosine diphosphate (ADP) in the microplate. Platelet aggregation was then measured every minute for 8 min. None of the estrogens (E1, E2 and E3) affected platelet aggregation in untreated PRP. Interestingly, only E1 and E3 can synergize the increased platelet aggregation by either ADR or ADP, while the effects of E2 on the increased platelet aggregation by either ADR or ADP depended on internal factors such as endogenous estradiol and platelet aggregated state. Thus, for the rational use of these internal factors for estrogen use, especially E2, in clinical applications, such as hormone replacement therapy, may need evaluation of thrombotic risk.

The expression of COX-2 in VEGF-treated endothelial cells is mediated through protein tyrosine kinase

Cyclooxygenase (COX), existing as the COX-1 and COX-2 isoforms, converts arachidonic acid to prostaglandin H2, which is then further metabolized to various prostaglandins. Vascular endothelial growth factor (VEGF) has been shown to play important roles in inflammation and is upregulated by the prostaglandin E series through COX-2 in several cell types. Here, we have investigated the effects of VEGF on the COX isoform expressed in human umbilical vein endothelial cells (HUVEC). The signalling mechanism of the COX isoform expressed in endothelial cells activated with VEGF will be also investigated using the tyrosine kinase inhibitor, genistein, and protein kinase C inhibitor, staurosporine. The activity of COX-2 was assessed by measuring the production of 6-keto-prostaglandin F1alpha in the presence of exogenous arachidonic acids (10 microM, 10 min) by enzyme immunoassay. The expression of COX isoform protein was detected by immunoblot using specific antibodies. Untreated HUVEC contained no COX-2 protein. In HUVEC treated with VEGF (0.01-50 ng/ml), COX-2 protein, but not COX-1, and COX activity were increased in a dose-dependent manner. Interestingly, the increased COX-2 protein and activity in response to VEGF (10 ng/ml) was inhibited by the tyrosine kinase inhibitor, genistein (0.05-5 microg/ml), but not by the protein kinase C inhibitor, staurosporine (0.1-10 ng/ml). Thus, the induction of COX-2 by VEGF in endothelial cells was mediated through protein tyrosine kinase, and the uses of specific COX-2 inhibitors in these conditions, in which VEGF was involved, might have a role.

The Effects of Thai Herbal Ha-Rak Formula on COX Isoform Expression in Human Umbilical Vein Endothelial Cells Induced by IL-1β

Objective To investigate the modulated effects of HRF on cyclooxygenase isoform expression and its activity, using the human umbilical vein endothelial cell (HUVEC) model induced by interleukin-1 beta (IL-1β). Methods Cells were treated with indomethacin (positive control), HRF, and its components at various concentrations prior to treatment with IL-1β at 24 h. Cell viability was determined by MTT assay. Moreover, the anti-inflammatory effects of HRF and its components through mRNA and protein expression were established using real-time quantitative PCR and Western blot, respectively. COX activity was identified via exogenous and endogenous PGE2 productions using the EIA. Result There was no cytotoxicity in HUVECs treated with HRF. None of the experimental conditions used in the study affected the expression of COX-1, but COX-2 protein expression was inhibited at concentrations under 10 µg/mL. Despite the significantly increased levels of exogenous PGE2, HRF had no effect on COX-2 mRNA expression. However, the production of PGE2 was lower at a concentration of 100 µg/mL HRF than at a concentration below 10 µg/mL. Interestingly, each component of HRF revealed different effects of the Ha-Rak formula. Conclusion Our preliminary findings suggest that HRF and its components provide diverse modulation of COX-2 and PGE2 at the in vitro level.