Name: BIANCA DE PAULA E RODRIGUES

Publication date: 21/12/2017
Advisor:

Name	Role
ELISARDO CORRAL VASQUEZ	Advisor *

Examining board:

Name	Role
ALESSANDRA SIMAO PADILHA	Internal Examiner *
BIANCA PRANDI CAMPAGNARO	External Examiner *
ELISARDO CORRAL VASQUEZ	Advisor *
FERNANDA GOBBI AMORIM	External Examiner *
SILVANA DOS SANTOS MEYRELLES	Internal Examiner *

Summary: A decline in the functionality of stem cells may be a key component in the pathogenesis of cardiovascular diseases. In atherosclerosis, there is an increase in endogenous genotoxic agents, such as reactive oxygen species (ROS), which can cause oxidative damage in DNA. Considering previous report that sildenafil, an inhibitor of phosphodiesterase 5 (PDE5), have antioxidant effects, in the present study we evaluated the effect of this drug on ROS levels, on pro-oxidant and antioxidant systems, on genotoxicity, on DNA repair kinetics and on apoptosis in hematopoietic stem cells (HSC) of atherosclerotic apoE knockout mice (apoE-/-). For this study were used male 20-week old apoE-/-
mice. Animals were distributed into three different groups: apoE−/− mice
administered with the PDE5 inhibitor sildenafil (apoE−/− sildenafil, Viagra®, 40 mg/kg/day, for 3 weeks, by oral gavage, n=25), apoE-/- mice administered with vehicle (apoE-/- vehicle, n=25) and Wild-type C57Black/6 mice (C57 vehicle, n=25). Then, animals were euthanized, blood collected for analysis of the lipid profile. The HSC were isolated by cell culture for assessed of ROS production, DNA damage, DNA repair kinetics and apoptosis by flow cytometry. Statistical comparisons were done by ANOVA, followed by Bonferroni’s post hoc test.
Values of *p<0.05 vs. C57 vehicle; #p<0.05 vs. apoE-/- vehicle and $p <0.05 vs. background. ApoE-/- vehicle group HSC showed an increase in intracellular ROS levels (1.3-fold) compared with control animals and administration of sildenafil decreased these levels (22%). It was observed a higher influence of NADPH oxidase and xanthine oxidase systems on ROS production in atherosclerotic animals, treatment with sildenafil was able to decrease this influence at levels similar to control. As well as, the antioxidant enzymes influence such as superoxide desmutase, catalase and glutathione peroxidase on the reduction of ROS levels was lower in the apoE-/- vehicle animals when compared with controls and the treatment with sildenafil increased this enzymes
influence in the reduction of ROS levels. Similarly, HSC from apoE-/- vehicle mice showed a 4-fold oxidative DNA fragmentation compared with C57 vehicle and sildenafil-administered apoE-/- mice exhibited minimal DNA damage in HSC similar to control mice. In addiction, a decrease in HSC’s DNA repair capacity was observed in apoE-/- vehicle animals when compared to C57 vehicle animals and treatment with sildenafil was able to increase this ability at levels similar to
controls animals. The results also showed an increase in the percentage of HSC in apoptosis in apoE-/- vehicle animals (7.0-fold) when compared to C57 vehicle animals and treatment with sildenafil was able to completely reverse this condition. Our data shows that atherosclerosis increases ROS production in HSC, apparently, increasing the pro-oxidant systems influence and decreasing antioxidant capacity. Thus, this imbalance in oxidative stress leads to an augment in genotoxicity, and, consequent, overload in the DNA repair system.
The higher levels of DNA fragmentation can be associated with increased apoptosis in HSC. Interestingly, the treatment with sildenafil was able to reduce oxidative stress and, consequently, oxidative damage and DNA repair overload, reducing apoptosis levels in HSC of atherosclerotic animals. The novelty of this study is that sildenafil may offer a new perspective to the use of PDE5 inhibitors
to protect against DNA damage, in cells involved in the organism plasticity in atherosclerosis.

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