Nuclear factor-kappa B, p38, and stress-activated protein kinase mitogen-activated protein kinase signaling pathways regulate proinflammatory cytokines and apoptosis in human placental explants in response to oxidative stress: effects of antioxidant vitamins

Abstract

Preeclampsia is a potentially fatal complication of human pregnancy characterized by hypertension, proteinuria, and edema. Placental oxidative stress is a key element in the pathogenesis of the syndrome and results in the release of a cocktail of factors, including proinflammatory cytokines and apoptotic debris, that in turn cause activation of the maternal endothelium. The intermediary molecular mechanisms underlying this release are unknown, but they represent a potential target for therapeutic interventions. We examined activation of signaling pathways during hypoxia-reoxygenation of villous explants in vitro. Hypoxia-reoxygenation activated the p38 and stress-activated protein kinase mitogen-activated protein kinase (MAPK) and the nuclear factor-kappaB pathways. Downstream consequences included increased tissue concentrations and secretion of tumor necrosis factor-alpha and interleukin-1 beta, increased expression of cyclooxygenase-2, and increased apoptosis. Administration of vitamins C and E to explants blocked activation of the p38 and stress-activated protein kinase MAPK and nuclear factor-kappaB pathways. Vitamin administration or p38 pathway inhibition also reduced cyclooxygenase-2 expression, tumor necrosis factor-alpha and interleukin-1 beta secretion, and the levels of apoptosis. We conclude that oxidative stress is a potent inducer of placental synthesis and release of proinflammatory factors. Most of these effects are mediated through the p38 MAPK and nuclear factor-kappaB pathways and can be effectively blocked by vitamins C and E in vitro.

Figure 1

In vitro H/R stimulates the expression of oxidative stress markers; simultaneous addition of vitamins suppresses the effects. Protein lysates from placentas cultured under H/R in the presence or absence of vitamins C and E (V) for 7 hours (n = 6) or 16 hours (n = 6) were analyzed for Hsp27 (a), Hsp90 (b), and HNE (c). Representative blots show the 7-hour time point. *P < 0.05 compared with normoxic control; ^#P < 0.05 compared with H/R treatment.

Figure 2

In vitro H/R activates p38 MAPK, SAPK, and NF-κB pathways; simultaneous addition of vitamins or a p38 inhibitor suppresses the effects of H/R. Protein lysates from placentas cultured under H/R in the presence or absence of vitamins C and E (V) or PD169316 (P-I) for 7 hours (n = 6) or 16 hours (n = 6) were analyzed for P-p38, p38 (a); P-SAPK, SAPK (b); and P-IκB, IκB (c). Representative blots show the 7-hour time point. *P < 0.05 compared with normoxic control; ^#P < 0.05 compared with H/R treatment.

Figure 3

In vitro H/R activates the tissue production and secretion of inflammatory cytokines and COX-2; the addition of vitamins or the p38 inhibitor suppresses the effects of H/R. a: Supernatants from placentas cultured under H/R in the presence or absence of vitamins C and E (V) or PD169316 (P-I) for 7 hours (n = 6) or 16 hours (n = 6) were analyzed by enzyme-linked immunosorbent assay for the secretion of TNF-α. Data were normalized against wet tissue weights. Corresponding protein lysates were analyzed with IL-1β (b) and COX-2 (c, d). d: Effect of the two p38 inhibitors, PD169316 (PD) and SB202190 (SB), was comparable. Representative blots show the 7-hour time point. *P < 0.05 compared with normoxic controls; ^#P < 0.05 compared with H/R.

Figure 4

In vitro H/R stimulates apoptosis; the addition of vitamins or the p38 inhibitor suppress the effects of H/R. Protein lysates from placentas cultured under H/R in the presence or absence of vitamins C and E (V) or PD169316 (P-I) for 7 hours (n = 6) or 16 hours (n = 6) were analyzed for cleaved caspase-3 (undetectable at 7 hours) (a) and cleaved caspase-9 (b). Representative blots show the 16-hour time point. *P < 0.05 compared with normoxic control; ^#P < 0.05 compared with H/R treatment.

Figure 5

Immunostaining against nitrotyrosine (a), HNE (b), Hsp27 (c), P-NF-κB (d), COX-2 (e), and cleaved caspase-9 and M30 (f) localized H/R-induced increase in these markers principally to trophoblast. Scale bar = 50 μm.