The major sources of superoxide anion formation and oxidative stress are NAD(P)H oxidase complexes in endothelial cells. Here we analyzed the expression of Nox2 and Nox4 isoformes in human endothelial cells and macrophages, studied the intracellular localization and determined the impact of overexpression of these Nox isoformes on endothelial superoxide anion formation.

In primary cultures of human umbilical artery or vein endothelial cells, we identified by high density oligonucleotide arrays containing 22.215 transcripts as major endothelial NAD(P)H oxidase subunits Nox4 and p22phox. All other subunits were below the level of detection. This expression pattern was confirmed by real time PCR (HUVEC: Nox4: 1.5±0.3 • 10 ⁴ RU, Nox2: 19±18 RU). The full-length coding regions of Nox2, Nox4 and p22phox cDNAs were cloned into the pcDNA4/TO vector and their identity was confirmed by DNA sequencing and Western blotting using the vector-specific myc-tag. Nox2-myc and Nox4-myc could be detected by immunofluorescence in the cytoskeleton of endothelial cells.

In contrast to Nox2, Nox4 overexpression resulted in augmented superoxide anion formation (282±53% RLU of control, P<0.05). In conclusion, Nox4 is the major Nox isoform of NAD(P)H oxidase in human endothelial cells in vitro.