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VASCULAR BIOLOGY

Nox4 NADPH oxidase mediates oxidative stress and apoptosis caused by TNF- in cerebral vascular endothelial cells

Laboratory for Research in Neonatal Physiology, Department of Physiology, University of Tennessee Health Science Center, Memphis, Tennessee

Submitted 21 July 2008 ; accepted in final form 30 December 2008

Inflammatory brain disease may damage cerebral vascular endothelium leading to cerebral blood flow dysregulation. The proinflammatory cytokine TNF- causes oxidative stress and apoptosis in cerebral microvascular endothelial cells (CMVEC) from newborn pigs. We investigated contribution of major cellular sources of reactive oxygen species to endothelial inflammatory response. Nitric oxide synthase and xanthine oxidase inhibitors (N-nitro-L-arginine and allopurinol) had no effect, while mitochondrial electron transport inhibitors (CCCP, 2-thenoyltrifluoroacetone, and rotenone) attenuated TNF--induced superoxide (O₂^–) and apoptosis. NADPH oxidase inhibitors (diphenylene iodonium and apocynin) greatly reduced TNF--evoked O₂^– generation and apoptosis. TNF- rapidly increased NADPH oxidase activity in CMVEC. Nox4, the cell-specific catalytic subunit of NADPH oxidase, is highly expressed in CMVEC, contributes to basal O₂^– production, and accounts for a burst of oxidative stress in response to TNF-. Nox4 small interfering RNA, but not Nox2, knockdown prevented oxidative stress and apoptosis caused by TNF- in CMVEC. Nox4 is colocalized with HO-2, the constitutive isoform of heme oxygenase (HO), which is critical for endothelial protection against TNF- toxicity. The products of HO activity, bilirubin and carbon monoxide (CO, as a CO-releasing molecule, CORM-A1), inhibited Nox4-generated O₂^– and apoptosis caused by TNF- stimulation. We conclude that Nox4 is the primary source of inflammation- and TNF--induced oxidative stress leading to apoptosis in brain endothelial cells. The ability of CO and bilirubin to combat TNF--induced oxidative stress by inhibiting Nox4 activity and/or by O₂^– scavenging, taken together with close intracellular compartmentalization of HO-2 and Nox4 in cerebral vascular endothelium, may contribute to HO-2 cytoprotection against inflammatory cerebrovascular disease.

inflammatory brain disease; brain endothelium; blood-brain barrier; cerebral vascular injury; heme oxygenase isoform-2; carbon monoxide-releasing molecule-A1; reduced nicotinamide adenine dinucleotide phosphatase oxidase isoforms 2 and 4; bilirubin; antioxidants