Primary role of superoxide anion generation in the cascade of events leading to endothelial dysfunction and damage in high glucose treated HUVEC.

Aim: The aim of the study was to elucidate the chain of events leading to oxidative damage in endothelial cells exposed to high glucose.

Method: The nitric oxide synthase (NOS) cofactor tetrahydrobiopterin (BH4), the peroxynitrite decomposition catalyst FP15, the inhibitor of mitochondrial complex II thenoyltrifluoroacetone (TTFA) and the antioxidant superoxide dismutase (SOD) mimetic Mn(III)tetrakis(4-benzoic acid) porphyrin chloride (MnTBAP) were individually added to human umbilical vein endothelial cells (HUVEC) cultured in high glucose. This study was designed to establish the possible sequence of action of NOS, peroxynitrite and superoxide anion in the oxidative damage cascade.

Results: We found that in high glucose, nitrotyrosine, 8OHdG, NO (+40%) and O2- (+300%) production, eNOS and caspase-3 expression increased, while Bcl-2 expression decreased. MnTBAP and TTFA were able to normalize all the parameters assayed. FP15 caused an increase in NO production, did not interfere with eNOS expression and O2- generation, but was able to reduce apoptosis and to normalize nitrotyrosine and 8OHdG formation. BH4 enrichment was able to reduce O2- generation, nitrotyrosine and 8OHdG formation and apoptosis. The addition of this cofactor did not affect eNOS expression, but increased NO formation, more than FP15.

Conclusion: These data show the starting role of superoxide anion generated at mitochondrial level in the cascade of events leading to hyperglycemia generated apoptosis.