Abrupt reoxygenation of microvascular endothelial cells after hypoxia activates ERK1/2 and JNK1, leading to NADPH oxidase-dependent oxidant production.

Objective: Mitogen-activated protein kinases (MAPK) in microvascular endothelial cells (EC) may participate in organ pathophysiology following hypoxia/reoxygenation (H/R). The authors aimed to determine the role of MAPK in H/R-induced reactive oxygen species (ROS) generation in mouse microvascular EC.

Methods: Cultured EC derived from skeletal muscle of male wild-type (WT), gp91phox-/- or p47phox-/- mice were subjected to hypoxia (0.1% O2, 1 h) followed by abrupt reoxygenation, H/RA (hypoxic medium quickly replaced by normoxic medium), or slow reoxygenation, H/RS (O2 diffused to cells through hypoxic medium). Cells were analyzed for ERK, JNK, and p38 MAPK phosphorylation, NADPH oxidase activation, and ROS generation.

Results: In WT cells, H/RA but not H/RS rapidly phosphorylated ERK1/2 and JNK1 and subsequently increased ROS production. H/RA did not affect p38. MAPK phosphorylation persisted despite inhibition of NADPH oxidase, mitochondrial respiration, protein tyrosine kinase, or PKC. ROS increase during H/RA was prevented by deletion of gp91phox or p47phox, or MAPK inhibition.

Conclusions: Abrupt reoxygenation after hypoxia activates ERK1/2 and JNK1 in mouse microvascular endothelial cells via a tyrosine kinase-, PKC-, and NADPH oxidase-insensitive mechanism, leading to increased NADPH oxidase-dependent ROS production. The results suggest that MAPK activation in the microvascular endothelium is O2-sensitive, contributing critically to tissue pathophysiology after H/R.