However, the current study suggests that sera from preeclamptic women contains factors capable of inducing NOX, which cannot be mitigated by hydroxychloroquine. Intriguingly, hydroxychloroquine was able to mitigate the effects of both TNF- and sera from preeclamptic women on the loss of endothelial ZO-1 and integrity. hypoxic injury or oxidative stress. Similarly, human umbilical vein endothelial cells (HUVECs) were used to assess the effects of hydroxychloroquine on in vitro markers of endothelial dysfunction. Hydroxychloroquine had no effect on the release of sFlt-1, sEng, TNF-, activin A, or 8-isoprostane from placental explants exposed to hypoxic injury or oxidative stress. However, hydroxychloroquine mitigated TNF–induced HUVEC production of 8-isoprostane and Nicotinanamide adenine dinucleotide phosphate (NADPH) oxidase expression. Hydroxychloroquine also mitigated TNF- and preeclamptic serum-induced HUVEC monolayer permeability and rescued the loss of zona occludens protein zona occludens 1 (ZO-1). Although hydroxychloroquine had no apparent effects on trophoblast function, it may be a useful endothelial protectant in women presenting with preeclampsia. = 0.02), sEng (Physique 1b, = 0.02), and TNF- (Physique 1c, = 0.02) from explant cultures after 24 h incubation. In the presence of X-XO (xanthine/xanthine oxidase system), explants cultured for 48 h significantly increased secretions of 8-isoprostane (Physique 2a, = 0.03) and activin A (Physique 2b, = 0.01) compared to controls. Co-incubation with 1 g/mL hydroxychloroquine did not alter either the hypoxia-induced secretion of sFlt-1 (Physique 1a), sEng (Physique 1b), or TNF- (Physique 1c), or the X-XO-induced increase in 8-isoprostane (Physique 2a) and activin A (Physique 2b). Open in a separate window Physique 1 Release of (a) soluble fms-like tyrosine kinase-1 (sFlt-1), (b) soluble endoglin (sEng), and (c) tumour necrosis factor- (TNF-) by placental explants of human term normal pregnancy placentae after 24 h incubation at 5% oxygen concentration (normoxia) versus 1% oxygen (hypoxia). The explants were incubated in the hypoxic environment in the absence or presence of 1 1 g/mL hydroxychloroquine. Data are mean standard error of the mean (SEM) from 10 impartial biological replicates. * denotes 0.05. NT: non treated, HCQ: hydroxychloroquine. Open in a separate window Physique 2 Release of (a) 8-isoprostane and (b) activin A by placental explants of human term normal pregnancy placentae after 48 h incubation at 20% oxygen concentration with 5% CO2. The explants were incubated in media made up of xanthine (2.3 mM) + xanthine oxidase (15 mU/mL) in the absence or presence of 1 1 g/mL hydroxychloroquine. Data are mean SEM from 10 impartial biological replicates. * denotes 0.05. X/XO: xanthine/xanthine oxidase, HCQ: hydroxychloroquine. 2.2. Effect of Hydroxychloroquine on HUVEC Viability Previously we have exhibited that, compared to untreated controls, there was no effect of hydroxychloroquine on human umbilical vein endothelial cell (HUVEC) viability across a dose range of 0.1, 1, and 10 g/mL over 120 h in culture [25]. However, treatment of cells with 100 g/mL Rabbit polyclonal to ZFP161 hydroxychloroquine significantly reduced cell viability at 24 h ( 0.001) [25]. Dosing of hydroxychloroquine for all those subsequent experiments were based on these results. 2.3. Effects of Hydroxychloroquine on Endothelial Function In Vitro HUVECs were treated in the absence or presence of (i) TNF- (100 ng/mL), (ii) sera from normal pregnancies (20%), or (iii) sera from preeclamptic women (20%) in the presence or absence of hydroxychloroquine (1 g/mL) to assess endothelial dysfunction (Physique 3). Compared to controls, incubation of HUVECs with TNF- (Physique 3a,c) or sera from preeclamptic women (Physique 3b,d) significantly increased both NADPH oxidase 2 (NOX2) mRNA expression ( 0.001 and = 0.01, respectively) and 8-isoprostane secretion (= 0.02 and = 0.04, respectively). Co-treatment of HUVECs with TNF- and hydroxychloroquine significantly reduced NOX2 mRNA expression (Physique 3a, = 0.03) and secretion of 8-isoprostane (Physique 3c, = 0.04). Co-treatment of HUVECs with serum from preeclamptic women and hydroxychloroquine did not significantly alter the expression of NOX2 mRNA or 8-isoprostane. However, 100 M apocynin, a NOX inhibitor, significantly reduced the NOX2 mRNA expression and 8-isoprostane release induced by serum from preeclamptic women (Physique 3b,d, respectively, 0.01 for both). Open in a separate window Physique 3 NADPH oxidase 2 (NOX2) RNA expression of human umbilical vein endothelial cells (HUVECs) treated with 100 ng/mL TNF- (a) and 20% preeclampsia (PE) sera (b). Release of 8-isoprostane by HUVECs treated with 100 ng/mL recombinant TNF- (c) and 20% preeclampsia sera (d). Data are mean SEM from eight impartial biological replicates. * denotes 0.05; ****p 0.001. Compared to controls, incubation of HUVECs with TNF- (Physique 4a) or 20% sera from preeclamptic women (Physique 4b) increased immunoreactivity for NOX2 protein. Once again, co-treatment of HUVECs with TNF- and either apocynin or hydroxychloroquine reduced immunoreactive NOX2 protein expression (Physique 4a). Similarly, co-treatment of HUVECs with sera from preeclamptic women and either apocynin or hydroxychloroquine also showed reduced immunoreactive NOX2 protein expression (Physique 4b). Open in a separate window Physique 4 Western blot representative for NOX2 protein.For all those in vitro experiments, 20% pooled sera from preeclampsia pregnancies were used for treatment of endothelial cells and were compared with that of the normotensive sera treated cells. serum-induced HUVEC monolayer permeability and rescued the loss of zona occludens protein zona occludens 1 (ZO-1). Although hydroxychloroquine had no apparent effects on trophoblast function, it may be a useful endothelial protectant in women presenting with preeclampsia. = 0.02), sEng (Physique 1b, = 0.02), and TNF- (Physique 1c, = 0.02) from explant cultures after 24 h incubation. In the presence of X-XO (xanthine/xanthine oxidase system), explants cultured for 48 h significantly increased secretions of 8-isoprostane (Physique 2a, = 0.03) and activin A (Physique 2b, = 0.01) compared to controls. Co-incubation with 1 g/mL hydroxychloroquine did not alter either the hypoxia-induced secretion of sFlt-1 (Physique 1a), sEng (Physique 1b), or TNF- (Physique 1c), or the X-XO-induced increase in 8-isoprostane (Physique 2a) and activin A (Physique 2b). Open in a separate window Physique 1 Release of (a) soluble fms-like tyrosine kinase-1 (sFlt-1), (b) soluble endoglin (sEng), and (c) tumour necrosis factor- (TNF-) by placental explants of human term normal pregnancy placentae after 24 h incubation Diphenhydramine hcl at 5% oxygen concentration (normoxia) versus 1% oxygen (hypoxia). The explants were incubated in the hypoxic environment in the absence or presence of 1 1 g/mL hydroxychloroquine. Data Diphenhydramine hcl are mean standard error of the mean (SEM) from 10 impartial biological replicates. * denotes 0.05. NT: non treated, HCQ: hydroxychloroquine. Open in a separate window Physique 2 Release of (a) 8-isoprostane and (b) activin A by placental explants of human term normal pregnancy placentae after 48 Diphenhydramine hcl h incubation at 20% oxygen concentration with 5% CO2. The explants were incubated in media made up of xanthine (2.3 mM) + xanthine oxidase (15 mU/mL) in the absence or presence of 1 1 g/mL hydroxychloroquine. Data are mean SEM from 10 impartial biological replicates. * denotes 0.05. X/XO: xanthine/xanthine oxidase, HCQ: hydroxychloroquine. 2.2. Effect of Hydroxychloroquine on HUVEC Viability Previously we have demonstrated that, compared to untreated controls, there was no effect of hydroxychloroquine on human umbilical vein endothelial cell (HUVEC) viability across a dose range of 0.1, 1, and 10 g/mL over 120 h in culture [25]. However, treatment of cells with 100 g/mL hydroxychloroquine significantly reduced cell viability at 24 h ( 0.001) [25]. Dosing of hydroxychloroquine for all those subsequent experiments were based on these results. 2.3. Effects of Hydroxychloroquine on Endothelial Function In Vitro HUVECs were treated in the absence or presence of (i) TNF- (100 ng/mL), (ii) sera from normal pregnancies (20%), or (iii) sera from preeclamptic women (20%) in the presence or absence of hydroxychloroquine (1 g/mL) to assess endothelial dysfunction (Physique 3). Compared to controls, incubation of HUVECs with TNF- (Physique 3a,c) or sera from preeclamptic women (Physique 3b,d) significantly increased both NADPH oxidase 2 (NOX2) mRNA expression ( 0.001 and = 0.01, respectively) and 8-isoprostane secretion (= 0.02 and = 0.04, respectively). Co-treatment of HUVECs with TNF- and hydroxychloroquine significantly reduced NOX2 mRNA expression (Physique 3a, = 0.03) and secretion of 8-isoprostane (Physique 3c, = 0.04). Co-treatment of HUVECs with serum from preeclamptic women and hydroxychloroquine did not significantly alter the expression of NOX2 mRNA or 8-isoprostane. However, 100 M apocynin, a NOX inhibitor, significantly reduced the NOX2 mRNA expression and 8-isoprostane release induced by serum from preeclamptic women (Physique 3b,d, respectively, 0.01 for both). Open in a separate window Physique 3 NADPH oxidase 2 (NOX2) RNA expression of human umbilical vein endothelial cells (HUVECs) treated with 100 ng/mL TNF- (a) and 20% preeclampsia (PE) sera (b). Release of 8-isoprostane by HUVECs treated with 100 ng/mL recombinant TNF- (c) and 20% preeclampsia sera (d). Data are mean .