Ogen-induced ROS in endothelial cells. DCF intensity was measured in endothelial cells co-treated with antioxidants

Ogen-induced ROS in endothelial cells. DCF intensity was measured in endothelial cells co-treated with antioxidants to verify E2-induced oxidant production. Human umbilical vein endothelial cells pretreated with the antioxidant DS5565 biological activity N-acetylcysteine (NAC) showed a significant reduction of E2-induced oxidants to the level of control. Data from three independent experiments are presented as ROS production with controls set at 100 (?SD). Values that are significantly different from E2 treatment alone (P < 0.05) are indicated with an asterisk (*).NA C((page number not for citation purposes)0m M) +0mPage 3 ofE2 (3 .EEE7pmol)BMC Cardiovascular Disorders 2006, 6:http://www.biomedcentral.com/1471-2261/6/150 100 50CT RL (3 67 pm ol ) fm ol)** ** **Figure 3 estrogen-induced reactive oxygen species Glutathione peroxidase mimic inhibits the formation of Glutathione peroxidase mimic inhibits the formation of estrogen-induced reactive oxygen species. Pretreatment with the glutathione peroxidase mimic ebselen showed a significant reduction of E2-induced oxidants in endothelial cells. Data from three independent experiments are presented as ROS production with controls set at 100 (?SD). PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28506461 Values that are significantly different from E2 treatment alone (P < 0.05) are indicated with an asterisk (*).G0-arrested estrogen dependent cells [17]. Therefore, we tested the influence of antioxidants on E2-induced DNA synthesis in vascular endothelial cells which is not considered to be an estrogen dependent tissue. E2-induced DNA synthesis at 18 h was evaluated by BrdU incorporation. E2 produced a significant 2-fold induction of BrdU incorporation (Figure 4). The similar increase was not observed in cells exposed to vehicle. We also evaluated the influence of the glutathione peroxidase mimic ebselen on E2induced DNA synthesis. Cotreatment with ebselen (20 ) significantly inhibited E2-induced DNA synthesis compared to E2 alone. This inhibitory effect was shown to be dose-dependent and suppressed E2-induced DNA synthesis by as much as 60 (Figure 4). The co-treatment of antioxidant NAC (1 mM) significantly decreased E2induced DNA synthesis by as much as 100 (Figure 5). The NAC (1 mM) cotreatment with E2, which is equal to the basal control level, did not inhibit DNA synthesis which showed that NAC completely counteracted the E2induced BrdU incorporation without affecting the basal levels of DNA synthesis. The inhibitory effect of NAC on E2-induced DNA synthesis was shown to be dose dependent.ROS Production( of control)EE(3 .DiscussionUntil recently, only nuclear ER signaling has been considered to be the major mechanism for regulating the growth of endothelial cells. High concentrations of E2 (10 ) have been shown to act as antioxidants in vitro [18]. In contrast, our study used physiological concentrations of E2 (367 fmol and 3.67 pmol per ml medium) which do not act as antioxidants. Here we present data leading to the major novel findings that: (i) physiological concentrations of E2 trigger a rapid production of intracellular ROS in endothelial cells and (ii) E2-induced DNA synthesis is mediated by ROS signaling in endothelial cells. In our model, cells were blocked at the G1/S phase boundary by serum starvation and then pushed into S phase by the addition of estrogen. We demonstrated that the antioxidants ebselen and NAC block E2-induced DNA synthesis or S phase progression. Like several growth factors such as platelet-derived growth factor, epidermal growth fact.