The PARP enzyme family consists of PARP-1 and several other poly(ADP-ribosyl)ating enzymes (26
). The key role of reactive oxygen and nitrogen species and resulting DNA single-strand breakage in PARP activation is supported by 1
) in vitro demonstration of an ~500-fold stimulation of ADP-ribose polymer synthesis after PARP binding to the broken DNA ends (28
) and 2
) numerous findings in cell culture and animal models of cardiovascular and neurodegenerative diseases, cancer, and inflammation as well as diabetes, suggesting that PARP activation is prevented or reversed by superoxide dismutase mimetics (26
), hydroxyl radical scavengers (26
), and peroxynitrite decomposition catalysts (3
), as well as indirect antioxidants, i.e., aldose reductase inhibitors (11
) or an-giotensin-2 converting enzyme inhibitors (31
). However, as discussed in the introduction, recent findings in cell culture models of diabetes suggest that early high-glucose–induced PARP activation could be mediated via some unidentified metabolic mechanism, different from oxidative-nitrosative stress, and contribute to rather than result from reactive oxygen and nitrogen species generation. The present study reveals the major contribution of PARP activation to nitrotyrosine formation in peripheral nerve, superoxide, and nitrotyrosine formation in epineurial arterioles of vasa nervorum and superoxide production in aorta of STZ-induced diabetic rats. Similar interactions between PARP activity and superoxide, HNE adduct, and nitrosylated protein abundance have been observed in high-glucose–exposed HSC. Note that PARP-1 abundance was not affected by high glucose or ABA treatment, but ABA decreased high-glucose–induced poly(ADP-ribosy-l)ated protein overexpression. The latter is consistent with the current view on PARP-1 as abundantly expressed in most cell types with very minor, if any, transcriptional regulation (26
A profound effect of the PARP inhibitor ABA on diabetes-associated oxidative-nitrosative stress, i.e., complete or essential correction of superoxide and nitrotyrosine production, cannot be attributed to weak antioxidant properties of the compound (32
) for two major reasons. First, the antioxidant properties of ABA are related to its ability to act as a hydroxyl radical scavenger and thus preserve intracellular reduced glutathione (32
). Such effect, observed with extremely high doses of the compound (500 mg · kg−1
i.p., i.e., >16-fold higher than in the present study, ), should lead to neuralization of hydrogen peroxide and prevent formation of lipid peroxide without any impact on superoxide formation. Superoxide is the source, but not the product, of hydrogen peroxide or hydroxyl radicals, and benzamides and related compounds (e.g., nicotinamide) do have superoxide anion radical scavenging properties. Second, similar effects of PARP inhibition on superoxide in aorta, superoxide and nitrotyrosine in vasa nervorum, and nitrosylated protein abundance in high-glucose–exposed HSC were also observed in our recent studies (34
) with another PARP inhibitor 1,5-isoquinolinediol employed at 10-fold (in vivo) and 250-fold (in vitro) lower doses than ABA. Such doses are unlikely to cause any direct antioxidant effects. Note that ABA’s effect on HNE adduct abundance, i.e., variable of lipid peroxidation can be attributed to both direct antioxidant properties of the compound and its independent effects on superoxide and nitrotyrosine formation.
PARP activation can contribute to diabetes-induced superoxide anion radical formation via multiple mechanisms. On the one hand, diabetes-associated decrease of peripheral nerve free mitochondrial and cytosolic NAD+
-to-NADH ratios is PARP-mediated and is reversed by a PARP inhibitor (35
). Therefore, PARP activation is likely to upregulate activities of both extramitochondrial NAD(P)H oxidase and mitochondrial NADH oxidase, the important superoxide-generating enzymes. Poly(ADP-ribosyl)ation of glyceraldehyde 3-phosphate dehydrogenase, if present, may lead to protein kinase C activation and advanced glycation end product formation (19
), both known to contribute to superoxide anion radical generation via protein kinase C–dependent activation of NAD(P)H oxidase (36
), Maillard reaction (37
), and advanced glycation end product interactions with their receptors (38
), as well as glycation and downregulation of antioxidative defense enzymes (39
). Thus, PARP activation may be involved in superoxide generation via multiple metabolic mechanisms. On the other hand, PARP activation alters transcriptional regulation and activates nuclear factor-κB, activator protein-1, signal transducer and activator of transcription-1, and others (18
). Such activation leads to increased formation of endothelin-1 (21
) and inflammatory cytokines, i.e., tumor necrosis factor-α, interleukin-6, and interleukin-1β1
, all known to contribute to superoxide generation (40
). In addition, PARP activation results in poly(ADP-ribosyl)ation of numerous mitochondrial proteins (42
), and the impact of this phenomenon on mitochondrial superoxide production remains to be explored.
Our finding of markedly reduced nitrotyrosine immunoreactivities in ABA-treated diabetic rats compared with the untreated diabetic group suggests that PARP activation is an important contributor to nitrosative stress in peripheral nerve and vasa nervorum in the STZ-induced diabetic rat model. Furthermore, quantitatively identical high-glucose–induced accumulation of nitrosylated (2.3-fold) and poly-(ADP-ribosyl)ated (1.8-fold) proteins in HSC and its complete or almost complete prevention by ABA treatment indicates that a similar mechanism operates in early human PDN. Nitrotyrosine is a footprint of peroxynitrite, a potent oxidant produced in the superoxide anion radical reaction with nitric oxide. Any approach counteracting superoxide formation, including PARP inhibition, will decrease peroxynitrite generation and nitrosylated protein abundance. It is important to remember that PARP activation leads to nuclear factor-κB–mediated upregulation of the iNOS gene (18
). iNOS is the main donor of nitric oxide for peroxynitrite formation in tissue sites for diabetes complications (45
). iNOS expression was increased in HSC cultured in 30 mmol/l glucose, compared with those cultured in 5.5 mmol/l glucose, and this increase was prevented by a PARP inhibitor treatment. Thus, PARP inhibition counteracts nitrosative stress via arrest of both superoxide and nitric oxide formation.
In addition to superoxide anion radicals and peroxynitrite, PARP activation may contribute to other free radical and oxidant formation in tissue sites for diabetes complications. It is reasonable to expect that by upregulating cyclooxygenase-2 (18
) and promoting glutamate accumulation (36
), PARP activation leads to increased production of hydroxyl radicals and hydrogen peroxide as well as lipid peroxidation (45
). The effect of ABA on HNE adduct abundance in high-glucose–exposed HSC in our study may be at least partially mediated via inhibition of COX-2 protein expression. The consequences of PARP activation in diabetic tissues cannot be completely understood without identification of poly (ADP-ribosyl)ated transcription factors and other extramitochondrial and intramitochondrial proteins, as well as PARP-regulated genes that encode factors involved in oxidant generation and antioxidative defense. Note that PARP activation contributes to Erk1/2 and p
-38 mitogen-activated protein kinase phosphorylation as well as inhibition of phosphatidyl inositol 3′-kinase/Akt pathway (47
), which makes the relations among oxidative-nitrosative stress, PARP activation, and a variety of metabolic and signaling pathways that can indirectly affect both oxidative stress and PARP even more complex.
In conclusion, PARP activation leads to oxidative-nitrosative stress in experimental PDN and high-glucose–exposed HSC. This probably occurs via multiple metabolic pathways and changes in gene expression. A complete understanding of the complex relations between oxidative-nitrosative stress and PARP activation in diabetes may require cutting-edge technologies such as genomics and proteomics.