Poly(ADP-ribose)Polymerase 1 Gene Deletion Protects Against Age-Dependent Endothelial Dysfunction

Background:Recent studies demonstrated that aging-associated endothelial dysfunction is related with the overproduction of reactive oxygen species (ROS), reactive nitrogen species (RNS) and decreased bioavailability of nitric oxide (NO). ROS is a pathophysiologically relevant endogenous trigger of DNA single-strand breakage and activation of PARP-1. Sustained DNA damage caused by oxidative stress can lead to the excessive activation of PARP-1.Recent experiments indicated that PARP-1 take part in the inflammatory response. Over-activation of PARP-1 represents an important mechanism of tissue damage and contributes to endothelial dysfunction in various pathological conditions associated with oxidative stress.. Studies of PARP-1 and age dependent endothelial dysfunction have not been reported. Our research using the natural aging mice to explore the role of PARP-1 inhibitor in protecting age-dependent endothelial dysfunction.Objectives:Explore the effect and mechanism of age-dependent endothelial function after inhibiting the expression of PARP-1.Methods:1. AnimalsWT and PARP-/- male mice, purchased from Jackson Laboratory (Bar Harbor, ME, USA) were divided into young and old groups (n=20 each group) respectively by age: 2 months (young) and 12 months (old). Then the biochemical criteria were measured.2.Measurement of vascular reactivityAortas were cut into 3-4 mm width rings. Each ring was connected to an isometric force transducer (JH-2B, Beijing, China).Vessel rings were preconstricted with norepnephrine, and their responses to acetylcholine and sodium nitroglycerin were recorded.3.RT-PCRRNA was extracted from aortas and cDNA was generated by standard methods. The mRNA level of β-actin, PARP-1, eNOS, iNOS, Arg2 were measured.4.Cell culture and treatmentHuman aortic endothelial cells were cultured in endothelial cell medium. AngⅡ was added into the culture medium of cells at 10-6 mol/L for 48 h. We used LY294002, BAY11-7082, and DPQ to inhibit the activity of Akt, NF-κB and PARP-1, respectively. SA-β-gal staining was used to detect the aged cells.5.Western blot analysisProtein was extracted from aortas and the cells and the expression of PARP-1, p-eNOS, eNOS, iNOS, AKT, p-AKT, p16, p21 was measured.6.Measurement of NO and superoxide productionNO content in plasma was assayed by use of the Total Nitric Oxide Assay Kit. Aortic and cells’ O2- production was assessed by using DHE and DCFH-DA.7. ImmunofluorescenceNuclear transition of NF-κB was measured by immunofluorescence. 8. Data are expressed as mean±SD. SPSS v12.0 for Windows (SPSS, Chicago, IL) was used for analysis. P< 0.05 was considered statistically significantResults:1.Age-dependent PARP-1 Expression in WT micePARP-1 protein level was 3.57-fold higher with aging (P<0.05). PARP-/- male mice express no PARP1.2. Age-associated alteration of vascular functionCompared with old WT mice, old PARP-1-/- mice showed restored endothelial-dependent relaxation to Ach (P<0.05). Endothelial-dependent relaxation was similar in young PARP-1-/- and WT mice to Ach (P>0.05). The response to the endothelial-independent vasodilator (Sodium Nitroprusside SNP) did not differ in young and old WT and PARP-1-/-mice (P>0.05, Figure 1E).3. Age-dependent changes in NO content and releaseCompared with young WT mice, NO and ROS levels significantly decreased in age WT mice, but PARP-1-/- can improve this change. eNOS, p-enos(Thr454), iNOS and Arg2 expression of aortic tissueCompared with young WT mice, the iNOS and Arg2 expressions of old WT mice were increased, but PARP-1-/- can improve this change. Then we measured the level of p-enos(Thr454) because of no change of eNOS level. Compared with young WT mice, the p-enos(Thr454) expression of old WT mice decreased, but PARP-1-/- can also improve this change.4. Aging cells and the expression of PARP-1Aging cells induced by AngⅡ was detected via SA-β-gal staining P16 and P21. And the expression of SA-B-gal staining P16 and P21 increased. Similarly, PARP-1 expression also increased.5.Level of ROS and NO in HAECAngll could enhanced ROS and decrease NO content.6.AngⅡ increased PARP-1 expression in HAECsCompared with AngⅡ-group, PARP-1 expression decreased after stimulation of AKT inhibitor.8. Inhibition of PARP-1 or NF-κB attenuated iNOS expression and increased P-enos(Thr454) expression but had no effect on eNOSCompared with AngⅡ-group, iNOS expression decreased after stimulation of AngII+PARP-1 inhibitor or AngII+NF-KB inhibitor, but it had no effect on eNOS. Then P-enos(Thr454) level decreased in AngⅡ-group, but AngII+PARP-1 inhibitor or AngII+NF-κB inhibitor could improve the change.9. PARP-1 inhibition reduced AngⅡ-induced iNOS expression by inhibiting NF-κB nuclear translocationNF-κB quickly transferred into the nucleus with AngⅡ stimulation. With PARP-1 or NF-κB inhibition, NF-κB remained in the cytoplasm.Conclusion:1. PARP-1 inhibition could protect against age-dependent endothelial dysfunction possibly by regulating NO bioavailability via iNOS.2.Inhibition of PARP-1 might help in treatment of vascular aging.