Antioxidant treatment prevents serum deprivation- and TNF-α-induced endothelial dysfunction through the inhibition of NADPH oxidase 4 and the restoration of β-oxidation.
OBJECTIVE
Oxidative stress plays a pivotal role in the impaired endothelial function occurring in vascular diseases. Antioxidant strategies induce a clinical advantage in patients with endothelial dysfunction and atherosclerosis and protect from oxidative damage, but the underlying molecular mechanisms have been poorly evaluated. The aim of this study was to analyze the effects and mechanisms of action of antioxidant regimens on endothelial function.


RESULTS
Antioxidant efficacy of N-acetylcysteine, ascorbic acid and propionyl-L-carnitine was evaluated in serum-deprived and TNF-α-stimulated human umbilical vein endothelial cells in vitro. Cell adhesion molecule (CAM) expression was evaluated by blot and real-time PCR, and inflammatory cytokine secretion was evaluated by ELISA; leukocyte adhesion and reactive oxygen species assays and NADPH oxidase 4 isoform (Nox4) expression analyses by blots were also performed. Antioxidant pretreatment restored serum-deprived and TNF-α-induced impaired mitochondrial β-oxidation by reducing flavin adenine dinucleotide level and counteracting increased CAM and Nox4 expression, leukocyte adhesion and inflammatory cytokine secretion. Specific inhibition by plumbagin and siNox4 prevented TNF-α- and serum deprivation-induced detrimental effects, confirming that endothelial oxidative stress and inflammation were Nox4 dependent.


CONCLUSIONS
Our findings documented Nox4 as a main actor in oxidative stress-induced endothelial dysfunction and further clarify the molecular basis of antioxidant treatment efficacy.
Department of Biomedicine and Prevention, Institute of Anatomic Pathology, University of Rome Tor Vergata, Rome, Italy.
Maria Giovanna
MG
Scioli
Alessandra
A
Bielli
Sara
S
Agostinelli
Chiara
C
Tarquini
Gaetano
G
Arcuri
Amedeo
A
Ferlosio
Gaetana
G
Costanza
Elena
E
Doldo
Augusto
A
Orlandi
D000111:Acetylcysteine / Q000494:pharmacology; D000975:Antioxidants / Q000494:pharmacology*; D001205:Ascorbic Acid / Q000494:pharmacology; D002331:Carnitine / Q000031:analogs & derivatives / Q000494:pharmacology; D002448:Cell Adhesion / Q000187:drug effects; D015815:Cell Adhesion Molecules / Q000378:metabolism; D002478:Cells, Cultured; D018920:Coculture Techniques; D016207:Cytokines / Q000378:metabolism; D019610:Cytoprotection; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D061307:Human Umbilical Vein Endothelial Cells / Q000187:drug effects* / Q000201:enzymology; D006801:Humans; D007962:Leukocytes / Q000187:drug effects / Q000378:metabolism; D008928:Mitochondria / Q000187:drug effects / Q000201:enzymology; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D009285:Naphthoquinones / Q000494:pharmacology; D010084:Oxidation-Reduction; D018384:Oxidative Stress / Q000187:drug effects*; D034622:RNA Interference; D044967:Serum / Q000378:metabolism*; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000633:toxicity*
D000975:Antioxidants; D015815:Cell Adhesion Molecules; D016207:Cytokines; D004791:Enzyme Inhibitors; D009285:Naphthoquinones; D014409:Tumor Necrosis Factor-alpha; C003223:propionylcarnitine; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C498910:NOX4 protein, human; D001205:Ascorbic Acid; D002331:Carnitine; D000111:Acetylcysteine; C014758:plumbagin