NADPH oxidase 5 as a driver of human umbilical artery dysfunction in preeclampsia

The objective of this study was to determine whether TNF-α–dependent activation of Nox5 contributes to endothelial dysfunction and vascular hyperreactivity associated with pre-eclampsia (PE). We hypothesized that increased TNF-α levels in PE promote intracellular Ca 2 + influx, leading to Nox5 activation, enhanced reactive oxygen species (ROS) generation, reduced nitric oxide (NO) bioavailability, and increased vascular contractility. Peripheral venous blood and umbilical cord arteries were collected from normotensive (NT) and PE pregnant women. Cytokine levels were determined by ELISA. Endothelial cells (EA.hy926) were stimulated with serum from NT or PE pregnancies (20% v/v, 1 h), in the presence or absence of infliximab (1 μM), nifedipine (1 μM), or Melittin (0.01 μM). Ca 2 + influx was assessed using a FLIPR probe. ROS generation was evaluated by lucigenin and Amplex Red assays. NO levels were determined using the Griess assay. Nox5 expression was reduced using siRNA. Umbilical artery rings were incubated with NT or PE serum, and concentration–response curves to serotonin were performed. Serum from PE pregnancies showed increased levels of TNF-α and IL-6 (pg/mL; TNF-α: NT 5.6±0.3 vs. PE 19.1±0.5; IL-6: NT 3.2±0.3 vs. PE 16.1±0.7). In endothelial cells, stimulation with PE serum and recombinant TNF-α significantly increased Ca 2 + influx (relative fluorescence units; NT 532±110 vs. PE 4568±134; vehicle 273±56 vs. TNF-α 2846±417). Pretreatment with infliximab partially inhibited this response (2626±127), whereas pretreatment with nifedipine completely abolished it (207±66). PE serum promoted increased O 2 - generation (relative chemiluminescence units; NT 92±19 vs. PE 751±26) and H 2 O 2 production (relative fluorescence units; NT 8.6±0.4 vs. PE 22.5±0.9). Pretreatment with infliximab partially reduced these increases (478±23), while nifedipine and Melittin completely abolished them (109±16 and 132±18, respectively). Basal NO production in PE serum–stimulated cells was reduced (nmol; NT 23.5±0.7 vs. PE 7.0±0.5). Infliximab partially prevented this reduction (15.6±0.6), whereas nifedipine and Melittin completely abolished it (21.7±0.5 and 22.0±0.9, respectively). Stimulation with PE serum significantly increased Nox5 gene expression in endothelial cells (2 - ΔCt; NT 1.7±0.01 vs. PE 5.8±0.1), an effect abolished by infliximab pretreatment (2.4±0.1). Nox5 gene silencing reduced Nox5 protein expression and prevented both ROS overproduction and NO reduction. Umbilical arteries from PE pregnancies exhibited higher Nox5 gene and protein expression (chemiluminescence intensity; NT 0.10±0.02 vs. PE 0.15±0.01), increased O 2 - generation (NT 742±121 vs. PE 2752±201) and H 2 O 2 production (NT 16.3±0.9 vs. PE 45.2±0.8), as well as reduced NO levels (nmol; NT 26.2±0.4 vs. PE 14.2±0.8), compared with arteries from NT pregnancies. Serotonin-induced contractile responses were greater in umbilical arteries stimulated with PE serum than with NT serum (Emax, g; NT 2.7±0.03 vs. PE 3.3±0.01). TNF-α–mediated Ca 2 + influx promotes Nox5 activation, leading to oxidative stress, reduced NO bioavailability, and increased vascular reactivity in pre-eclampsia. These findings identify Nox5 as a key molecular mediator of endothelial and vascular dysfunction associated with PE. Funding: FAPEG. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.