Skip to main content

Sildenafil restores endothelial function in the apolipoprotein E knockout mouse



Atherosclerosis is an inflammatory process of the arterial walls and is initiated by endothelial dysfunction accompanied by an imbalance in the production of reactive oxygen species (ROS) and nitric oxide (NO). Sildenafil, a selective phosphodiesterase-5 (PDE5) inhibitor used for erectile dysfunction, exerts its cardiovascular effects by enhancing the effects of NO. The aim of this study was to investigate the influence of sildenafil on endothelial function and atherosclerosis progression in apolipoprotein E knockout (apoE−/−) mice.


ApoE−/− mice treated with sildenafil (Viagra®, 40 mg/kg/day, for 3 weeks, by oral gavage) were compared to the untreated apoE−/− and the wild-type (WT) mice.

Aortic rings were used to evaluate the relaxation responses to acetylcholine (ACh) in all of the groups. In a separate set of experiments, the roles of NO and ROS in the relaxation response to ACh were evaluated by incubating the aortic rings with L-NAME (NO synthase inhibitor) or apocynin (NADPH oxidase inhibitor). In addition, the atherosclerotic lesions were quantified and superoxide production was assessed.


Sildenafil restored the vasodilator response to acetylcholine (ACh) in the aortic rings of the apoE−/− mice. Treatment with L-NAME abolished the vasodilator responses to ACh in all three groups of mice and revealed an augmented participation of NO in the endothelium-dependent vasodilation in the sildenafil-treated animals. The normalized endothelial function in sildenafil-treated apoE−/− mice was unaffected by apocynin highlighting the low levels of ROS production in these animals. Moreover, morphological analysis showed that sildenafil treatment caused approximately a 40% decrease in plaque deposition in the aorta.


This is the first study demonstrating the beneficial effects of chronic treatment with sildenafil on endothelial dysfunction and atherosclerosis in a model of spontaneous hypercholesterolemia. These data indicate that the main mechanism of the beneficial effect of sildenafil on the endothelial function appears to involve an enhancement of the NO pathway along with a reduction in oxidative stress.


Atherosclerosis, which can be defined as a chronic and progressive disease characterized by an inflammatory response of the arterial wall, is still a leading cause of death, mainly in the Western world [1, 2]. The development of atherosclerotic plaques is influenced by oxidative stress, an imbalance between the accumulation of reactive oxygen species (ROS) mainly produced by NADPH oxidase and limited antioxidant defenses; this imbalance compromises nitric oxide (NO) availability and leads to endothelial dysfunction [3, 4]. Early studies suggest that the correction of endothelial dysfunction results in a better prognosis [5], supporting the need for drugs to improve endothelial function and to reduce the progression of atherosclerosis [6].

Sildenafil, which has largely been used for erectile dysfunction and pulmonary hypertension [7], is being used to enhance in vivo NO signaling in other vascular diseases as well [6]. This drug increases the bioavailability of the intracellular second messenger of NO (cGMP) by inhibition of phosphodiesterase-5 (PDE5). Sildenafil was recently shown to improve endothelial function in coronary arterial disease as well as in diabetes and to reduce oxidative stress in many tissues [811]. However, the drug has not yet been tested in experimental models of atherosclerosis.

The apolipoprotein E knockout (apoE−/−) mouse developed two decades ago [12, 13], exhibits spontaneous hypercholesterolemia, accompanied by endothelial dysfunction similar to that observed in humans [4, 14, 15]. Therefore, the apoE−/− mouse seems to be a suitable genetic model to test new therapeutic approaches for the treatment of vascular diseases observed in dyslipidemias [16].

The aim of the present study was to test the hypothesis that chronic sildenafil treatment could revert or reduce the endothelial dysfunction and the progression of atherosclerosis observed in large vessels of apoE−/− mice. Our data show that this drug was able to restore the endothelial function in the aorta of apoE−/− mice, primarily by enhancing the activation of the NO pathway and by decreasing ROS production.

Materials and methods


Experiments were conducted in adult (18-week-old) male apoE−/− mice, which have a C57BL/6J genetic background, and on C57BL/6J wild-type (WT) mice, bred and maintained in the animal care facility at the Laboratory of Transgenes in the Health Sciences Center at the Federal University of Espirito Santo, Brazil. Mice were housed in individual plastic cages with a controlled temperature (22-23°C) and humidity (60%) and were exposed to a 12:12-h light–dark cycle. All experimental procedures were performed in accordance with the guidelines for the care and handling of laboratory animals as recommended by the National Institutes of Health (NIH), and study protocols were previously approved by the Institutional Animal Care Committee (CEUA-Emescam, Protocol # 007/2010).

To accelerate and aggravate the spontaneous hyperlipidemia and progression of endothelial dysfunction and atherosclerosis in apoE−/− mice, the animals were fed a Western-type diet for 10 weeks, starting at 8 weeks of age (AIN93G modified diet, Rhoster, Brazil). After 7 weeks (15-week-old), the mice were divided into three different groups: (a) apoE−/− mice treated with the PDE5 inhibitor sildenafil (Viagra®, 40 mg/kg/day, for 3 weeks, by oral gavage), (b) apoE−/− mice treated with the vehicle and (c) WT control mice, treated with the vehicle.

Measurement of plasma lipids

Blood samples were obtained by puncturing the right ventricle of the animals euthanized with an overdose of thiopental sodium anesthetic. The blood was immediately transferred to a tube containing heparin and centrifuged at 3000 RPM for 10 min. Plasma was separated immediately and kept at −20°C until assayed. Total plasma cholesterol, high density lipoproteins (HDL), low density lipoproteins (LDL) and triglycerides were determined using commercial colorimetric assay kits (Bioclin, Belo Horizonte, Brazil). Very low density lipoproteins (VLDL) and intermediate density lipoproteins (IDL) were estimated by subtracting HDL and LDL from total serum cholesterol.

Vascular function

After the treatment, animals were anesthetized with a sodium thiopental injection (200 mg/kg, i.p.), blood samples were collected and the thoracic aorta was removed and immediately placed in modified Krebs buffer of the following composition (in mM): NaCl 115, KCl 4.7, CaCl2·2H2O 2.5, MgSO4·7H2O 1.2, KH2PO4 1.2, NaHCO3 25, EDTA 0.01 and glucose 11.1. Loose connective tissue in the adventitia was carefully removed to avoid damage, and the vessel was cut into rings (3 to 4 mm thick). The aortic rings were then mounted on stainless steel triangles, suspended in vertical organ baths containing 5 ml Krebs solution, maintained at a resting tension of 0.5 g at 37°C, pH 7.4 and continuously gassed with a mixture of 95% O2 and 5% CO2. The rings were connected to force transducers to measure isometric tension through a computerized acquisition system (MP100, Biopac Systems Inc., Santa Barbara, USA).

After a 60-min equilibration period, aortic rings were exposed to KCl (125 mM) to assess their maximal tension. The endothelial function was then verified by acetylcholine (ACh)-induced relaxation (10 μM) after pre-contraction with phenylephrine (PE, 10 μM); the endothelium was considered functional when the relaxation was greater than 50%. After a washout period of 30 minutes, endothelium-dependent relaxation responses were tested by concentration-response curves to cumulative concentrations of ACh (100 pM – 30 μM).

In a separate set of experiments, the role of NO and ROS in the relaxation response to ACh were evaluated by incubation of the aortic rings with the non-specific NO synthase (NOS) inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME, 100 μM), or the NADPH oxidase inhibitor apocynin (300 μM) that were added to the vessel bath 20 minutes prior to assessing the concentration-response curves. The area under the curve (AUC) for each of the responses of the aortic rings to ACh in the presence or absence of L-NAME was calculated, and these results were expressed in arbitrary units.

Quantification of the atherosclerotic lesion

In another set of animals, the mice were euthanized with a sodium thiopental overdose (200 mg/kg, i.p.), and the left ventricle was perfused with 0.1 M phosphate-buffered saline (PBS, pH 7.4), followed by a 4% formaldehyde solution in PBS at a pressure of 100 mm Hg. The aortic root and a portion of the ascendant aorta were removed, placed in PBS, dissected free from fat and adhering perivascular tissue, embedded in OCT compound and cross-sectioned on a cryostat (Jung CM1800; Leica, Wetzlar, Germany) at a thickness of 7 μm. For each animal, aorta cross-sections were mounted on gelatin-coated slides and stained with Oil-Red-O (Sigma-Aldrich, St. Louis, MO, USA). Atherosclerotic lesions were quantified using a microscope (Olympus AX70, Olympus, Center Valley, PA, USA) interfaced with a video camera (VKC150, Hitachi, Tokyo, Japan) and an image analysis system (Image J, Public Domain). The lesion area per mouse was calculated by an investigator who was blinded to the experimental protocol and was expressed as a percentage of the lumen occupied by the atherosclerotic plaque.

Detection of superoxide production

Unfixed frozen sections from the aorta were cut into 7-μm-thick sections and mounted on gelatin coated glass slides. Samples were incubated with the oxidative fluorescent dye dihydroethidium (DHE, 2 μmol/L) in a modified Krebs's solution (containing 20 mM HEPES), in a light-protected humidified chamber at 37°C for 30 min, to detect superoxide. The intensity of fluorescence was quantified in the tissue sections using a microscope (Nikon Eclipse TI, Nikon Instruments Inc., Melville, NY, USA) by an investigator blinded to the experimental protocol.

Statistical analysis

Values were expressed as the means ± S.E.M. Relaxation responses to ACh and SNP were expressed as the percentage of dilation relative to the maximal pre-contraction level. For each concentration-response curve, the maximum effect (Rmax) and the log of the concentration of agonist that produced half of Rmax (log EC50) were calculated using nonlinear regression analysis. The sensitivities of the agonists were expressed as pEC50 (−log EC50). Statistical comparisons between the different groups were performed by one-way or two-way analysis of variance (ANOVA) followed by Bonferroni’s post hoc test. The statistical analyses were performed using Prism software (Prism 5, GraphPad Software, Inc., San Diego, CA, USA). A value of p < 0.05 was regarded as statistically significant.


Table 1 summarizes the results of plasma lipid profile of the three groups of animals. As expected, the apoE−/− vehicle group which received a Western type diet for 10 weeks, showed a significant increase in triglycerides (4-fold) along with a marked increase in total plasma cholesterol (14-fold) and VLDL + IDL (42-fold), when compared to the WT mice. On the other hand, HDL was significantly decreased (4-fold) in the apoE−/− mice compared to the WT mice. Sildenafil treatment did not change the profile in apoE−/− animals.

Table 1 Lipid profiles of vehicle- and sildenafil-treated apolipoprotein E-deficient (apoE−/−) mice fed a Western-type diet and wild-type control (WT)

The maximal vascular response (Rmax) and sensitivity (pEC50) to ACh were significantly decreased in the aortas of untreated apoE−/− animals, compared to those of the WT mice (Table 2, Figure 1A). This impaired ACh-induced relaxation response in the apoE−/− mice was completely restored on sildenafil treatment to values similar to those observed in the WT mice (Table 2, Figure 1B).

Table 2 Effect of L-NAME and apocynin on the efficacy of and sensitivity to acetylcholine in isolated aortic rings from wild-type control (WT) mice and from vehicle- and sildenafil-treated apolipoprotein E-deficient (apoE−/−) mice
Figure 1
figure 1

Effect of sildenafil treatment on the endothelial function of apoE−/− mice. (A) Acetylcholine (ACh)-induced endothelium-dependent relaxation in aortic rings, comparing untreated apoE−/− mice with wild-type (WT) control mice. (B) Effect of the treatment with sildenafil (40 mg/kg/day for 3 weeks) on ACh-induced vasodilation in apoE−/− mice compared to untreated apoE−/− and to WT mice. Values are the means ± SEM. *p < 0.05 vs. WT; #p < 0.05 vs. untreated apoE−/− (two-way ANOVA).

To evaluate the participation of NO in endothelium-dependent vasodilator responses, aortic rings were incubated with L-NAME prior to ACh concentration-response curves. In all groups, L-NAME did block the vasodilator response elicited by ACh (Table 2, Figures 2A, 2B, 2C). Moreover, analysis of the difference of the AUC revealed that the participation of NO in the relaxation response to ACh was significantly reduced in the untreated apoE−/− mice when compared to the WT mice (58 ± 17 vs. 230 ± 11, p < 0.01). Sildenafil treatment recovered the normal ACh-induced relaxation in apoE−/− mice when compared to untreated apoE−/− mice (233 ± 10, p < 0.01, Figure 2D).

Figure 2
figure 2

Participation of nitric oxide in ACh-induced vasodilation. Concentration-response curves show acetylcholine (ACh)-induced endothelium-dependent relaxation in aortic rings under the blockade of nitric oxide synthase with L-NAME in wild-type (WT) mice (A), untreated apoE−/− mice (B) and sildenafil-treated apoE−/− mice (C). Bar graph (D) shows the difference in the area under the curve (AUC) of the response of aortic rings to ACh with and without L-NAME. A decreased difference in the AUC indicates an impaired response to ACh. Values are the means ± SEM. **p < 0.01 vs. without L-NAME (A, B and C: two-way ANOVA); **p < 0.01 vs. WT and ##p < 0.01 vs. untreated apoE−/− (D: one-way ANOVA).

The participation of ROS in the relaxation response to ACh was assessed by incubation of the aortic rings with apocynin. We did not observe significant changes in the vascular reactivity after apocynin blockage in the WT animals, indicating that ROS may not participate in these responses (Table 2, Figure 3A). However, apocynin significantly increased maximal relaxing response and sensitivity to ACh in aortas of untreated apoE−/− mice (Table 2, Figure 3B). In apoE−/− mice treated with sildenafil, the ACh-induced vasorelaxation in the presence of apocynin was similar to that observed in WT mice (Table 2, Figure 3C).

Figure 3
figure 3

Participation of reactive oxygen species. Data show the effect of the blockade of NADPH oxidase activity with apocynin on the cumulative concentration–response curve to acetylcholine (ACh) in untreated and treated apoE−/− mice and wild-type (WT) control mice. Values are mean ± SEM. **p < 0.01 vs. apoE−/− without blockage (one-way ANOVA).

Figure 4 summarizes the results of histological analyses of aorta from the three groups of animals. ApoE−/− mice showed a significant plaque deposition in the aortic root when compared to WT animals (37.7 ± 3.4% vs. 0.0 ± 0.0%, p < 0.01). Sildenafil treatment in the apoE−/− animals caused approximately a 40% reduction of the atherosclerotic plaque deposition (21.3 ± 5.0, p < 0.05; Figure 4A). DHE staining revealed an increased oxidative stress in the aortas from untreated atherosclerotic apoE−/− animals (3.5 ± 0.4, p < 0.05) compared to the WT group (2.30 ± 0.1), and treatment with sildenafil reduced the oxidative stress in aortas from apoE−/− mice (2.4 ± 0.2, p < 0.05 vs. untreated apoE−/− mice; Figure 4B).

Figure 4
figure 4

Effect of sildenafil treatment on plaque deposition and oxidative stress. (A) Bar graph showing mean ± SEM values (in %) of plaque deposition in untreated and treated apoE−/− mice compared to wild-type (WT) control mice. Photomicrographs are typical aorta cross-sections of each group of animals. Scale bar 500 μm. (B) Bar graph showing mean ± SEM values of dihydroethidium (DHE) labeling (in arbitrary units) comparing the 3 groups of animals. Photomicrographs above the graph are typical aorta cross-sections showing fluorescent DHE labeling. Scale bar 100 μm. *p < 0.05 and **p < 0.01 untreated apoE−/− vs. WT; #p < 0.05 treated vs. untreated apoE−/− vehicle (one-way ANOVA).


This work showed for the first time that chronic inhibition of PDE5 with sildenafil restored endothelial function in the aorta of apoE−/− mice. In addition, sildenafil treatment reduced the atherosclerotic plaque deposition in this large vessel without affecting the plasma cholesterol levels. The amelioration of endothelial function and delayed progression of atherosclerosis that was observed, were mainly regulated through improving the basal NO pathway activation and decreasing ROS production.

As expected, the apoE−/− mice showed higher plasma cholesterol than the WT animals [1720], exhibiting approximately a 14-fold increase in total cholesterol, as recently reviewed [4]. Interestingly, sildenafil did not change the plasma lipid profile in the treated animals, which was consistent with similar results found elsewhere [21], although it improved the endothelial function. In recent years, there has been a significant improvement in the development of new therapeutics that target cardiovascular risk factors other than hypercholesterolemia [22]. Similar to the results in our present study, a class of statins has been shown to have pleiotropic effects with atheroprotective properties that were independent of cholesterol and were mediated by inhibition of isoprenoids [23]. Hence, we propose that the beneficial vascular effects of sildenafil may offer a new perspective for the use of PDE5 inhibitors in ameliorating endothelial dysfunction and reducing atherosclerosis.

It has been postulated that the major cause of endothelial dysfunction observed in apoE−/− mice is the reduction in the NO pathway, including the increased inactivation of this species by ROS [4, 24]. NO is a multifunctional molecule, exerting several potentially anti-atherosclerotic effects, such as antioxidant, anti-inflammatory, anti-platelet and vasodilator [4, 25]. Because the atherosclerotic disease is initiated by an endothelial dysfunction, novel therapeutics that potentiate or mimic the increase in NO bioavailability should also be considered. Sildenafil appears to be a promising alternative as it inhibits the degradation of cGMP, the second messenger of NO [4, 6, 26]. The potential advantage of a therapeutic approach based on inhibition of PDE5 is that, unlike strategies that attempt to increase NO levels (e.g., substrates, co-factors, antioxidants) [27], this one avoids risks associated with excessive NO. At high concentrations, NO is a cytotoxic free radical that increases oxidative stress independent of activation of guanylyl cyclase, thereby contributing to the development of atherosclerosis through overproduction of peroxynitrite (ONOO-) [28, 29].

In the present study, we observed marked endothelial dysfunction in the aorta of apoE−/− mice, as shown before by others in conductance and resistance vessels [24, 25, 30, 31]. We found that this endothelial dysfunction is related to reduced participation of NO in the vasodilator response to ACh, as demonstrated by a reduced area under the ACh concentration-response curve when blocked with L-NAME; this was in accordance with previous results [25]. It has already been shown that apoE−/− mice exhibit reduced activity of endothelial NO synthase (eNOS), even if the expression of the enzyme is normal [24, 32]. Furthermore, some studies have evaluated the hypothesis that the vascular dysfunction in apoE−/− mice may be dependent not only on the reduced bioavailability of NO but also on the altered responsiveness of vascular smooth muscle cells (VSMCs) to NO. We did not observe differences in sodium nitroprusside responses between WT and untreated apoE−/− mice (data not shown), demonstrating that the prejudice of vascular function is not due to a reduction in the sensitivity of smooth muscle cells to NO; this contradicts what was previously reported in the mesenteric arteriolar bed [33] and in the aorta [3436].

The efficacy of sildenafil in promoting the vasodilator response to ACh was demonstrated in diabetic rats [37] and in a secondhand smoke mouse model [38]; however, this efficacy has not yet been demonstrated in atherosclerotic experimental animals. The novel finding of our study is that sildenafil treatment restored endothelial function in this murine model of atherosclerosis. Once sildenafil main mechanism of action is inhibition of PDE5 in VSMCs, the drug mimics the increase in NO availability. In addition, recent data have shown that sildenafil can increase the expression [3941] and activity [28, 38] of eNOS, which could explain the amelioration of endothelial function and the reduction in atherosclerotic lesions.

Our data provide evidence that NADPH-derived ROS production contributes to oxidative stress in apoE−/− animals, because apocynin ameliorated ACh-induced relaxations in this group as shown previously [42]. The novelty of the present study is that sildenafil treatment restores endothelial function in apoE−/− mice by a reduction in oxidative stress. It has been shown that increased cGMP can inhibit NADPH oxidase expression/activity, thereby reducing ·O2- production, and consequently restoring NO bioavailability. This antioxidative effect of sildenafil may constitute an additional mechanism for the augmentation of NO-mediated relaxation [26]. These results on the oxidative stress, as assessed by a vascular reactivity study, are in accordance with the in situ observations using frozen sections labeled with DHE. In accordance with others [24, 43, 44], we observed that apoE−/− mice show increased DHE staining, which represents increased ·O2- production. Our finding is in agreement with another study, which demonstrated a reduction in nitrotyrosine immunostaining (a marker of local oxidative stress, produced by the interaction of ONOO- with tyrosine residues in peptides) in ischemic muscles of apoE−/− animals treated with sildenafil [21].

It has been postulated that atherogenesis is initiated with an endothelial dysfunction, in response to an injury to the endothelium caused by risk factors such as dyslipidemia [1, 4]. Thus, interventions that restore endothelial function can potentially reduce atherosclerotic plaque deposition. We observed that sildenafil treatment, in addition to amelioration of endothelial function in hypercholesterolemic apoE−/− mice, is capable of reducing atherosclerosis in the aorta of treated animals by approximately 40%. Atherosclerosis is considered an inflammatory disease, in which the migration of leucocytes through the endothelial wall is a crucial event. Circulating monocytes, attracted by chemoattractants that are released by activated endothelial cells, adhere to the vessel wall by interacting with E-selectin, vascular cell adhesion molecule (VCAM-1) and intercellular adhesion molecule (ICAM-1). This initiates a process of phagocytosis of modified LDL and foam cell formation [45]. Sildenafil can reduce atherosclerosis by interfering with this inflammatory cascade [11] by preventing oxidation of LDL, in addition to improving the endothelial function. A recent report has shown that a dose of sildenafil similar to that used in our study increases the number of bone marrow-derived endothelial progenitor cells in hypercholesterolemic apoE−/− mice [21]. These cells may be involved in the reduction of oxidative stress and apoptosis through cell therapy, as observed by our group recently [43],44], and this finding may offer new insight into similar effective therapies.

In conclusion, we are the first to show that chronic sildenafil treatment restores endothelial function and reduces oxidative stress in a murine atherosclerotic model based on a reduction in plaque deposition in these mice, independent of changes in the lipid profile. This study confirms that sildenafil is a promising novel pharmacologic strategy to treat atherosclerosis, although further studies are necessary to confirm the safety of this pharmacological approach in atherosclerotic patients.


  1. Ross R: Atherosclerosis - an inflammatory disease. N Engl J Med. 1999, 340: 115-126. 10.1056/NEJM199901143400207.

    Article  CAS  PubMed  Google Scholar 

  2. Libby P, Ridker PM, Hansson GK: Progress and challenges in translating the biology of atherosclerosis. Nature. 2011, 473: 317-325. 10.1038/nature10146.

    Article  CAS  PubMed  Google Scholar 

  3. De Meyer GRY, Herman AG: Vascular endothelial dysfunction. Prog Cardiovasc Dis. 1997, 39: 325-342. 10.1016/S0033-0620(97)80031-X.

    Article  CAS  PubMed  Google Scholar 

  4. Meyrelles SS, Peotta VA, Pereira TM, Vasquez EC: Endothelial dysfunction in the apolipoprotein E-deficient mouse: insights into the influence of diet, gender and aging. Lipids Health Dis. 2011, 10: 211-10.1186/1476-511X-10-211.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  5. Versari D, Daghini E, Virdis A, Ghiadoni L, Taddei S: Endothelial dysfunction as a target for prevention of cardiovascular disease. Diabetes Care. 2009, 32: S314-S321. 10.2337/dc09-S330.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  6. Tomasoni L, Sitia S, Borghi C, FG Ca, Ceconi C, Cecaro F, Morganti A, De Gennaro Colonna V, Guazzi M, Morricone L, Malavazos AE, Marino P, Cavallino C, Shoenfeld Y, Turiel M: Effects of treatment strategy on endothelial function. Autoimmun Rev. 2010, 9: 840-844. 10.1016/j.autrev.2010.07.017.

    Article  CAS  PubMed  Google Scholar 

  7. Palit V, Eardley I: An update on new oral PDE5 inhibitors for the treatment of erectile dysfunction. Nat Rev Urol. 2010, 7: 603-609. 10.1038/nrurol.2010.165.

    Article  CAS  PubMed  Google Scholar 

  8. Halcox JP, Nour KR, Zalos G, Mincemoyer RA, Waclawiw M, Rivera CE, Willie G, Ellahham S, Quyyumi AA: The effect of sildenafil on human vascular function, platelet activation, and myocardial ischemia. J Am Coll Cardiol. 2002, 40: 1232-1240. 10.1016/S0735-1097(02)02139-3.

    Article  CAS  PubMed  Google Scholar 

  9. Milani E, Nikfar S, Khorasani R, Zamani MJ, Mohammad A: Reduction of diabetes-induced oxidative stress by phosphodiesterase inhibitors in rats. Biochem Physiol C Toxicol Pharmacol. 2005, 140: 251-255. 10.1016/j.cca.2005.02.010.

    Article  Google Scholar 

  10. Shukla N, Jones R, Persad R, Angelini GD, Jeremy JY: Effect of sildenafil citrate and a nitric oxide donating sildenafil derivative, NCX 911, on cavernosal relaxation and superoxide formation in hypercholesterolaemic rabbits. Eur J Pharmacol. 2005, 517: 224-231. 10.1016/j.ejphar.2005.05.012.

    Article  CAS  PubMed  Google Scholar 

  11. Aversa A, Vitale C, Volterrani M, Fabbri A, Spera G, Fini M, Rosano GM: Chronic administration of Sildenafil improves markers of endothelial function in men with Type 2 diabetes. Diabet Med. 2008, 25: 37-44. 10.1111/j.1464-5491.2007.02298.x.

    Article  CAS  PubMed  Google Scholar 

  12. Plump AS, Smith JD, Hayek T, Aalto-Setälä K, Walsh A, Verstuyft JG, Rubin EM, Breslow JL: Severe hypercholesterolemia and atherosclerosis in apolipoprotein E-deficient mice created by homologous recombination in ES cells. Cell. 1992, 71: 343-353. 10.1016/0092-8674(92)90362-G.

    Article  CAS  PubMed  Google Scholar 

  13. Piedrahita JA, Zhang SH, Hagaman JR, Oliver PM, Maeda N: Generation of mice carrying a mutant apolipoprotein E gene inactivated by gene targeting in embryonic stem cells. Proc Natl Acad Sci USA. 1992, 89: 4471-4475. 10.1073/pnas.89.10.4471.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  14. Chiu JJ, Chien S: Effects of disturbed flow on vascular endothelium: pathophysiological basis and clinical perspectives. Physiol Rev. 2011, 91: 327-387. 10.1152/physrev.00047.2009.

    Article  PubMed  Google Scholar 

  15. Vasquez EC, Peotta VA, Gava AL, Pereira TMC, Meyrelles SS: Cardiac and vascular phenotypes in the apolipoprotein E-deficient mouse. J Biomed Sci. 2012, 19: 22-10.1186/1423-0127-19-22.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  16. Simonsen U, Rodriguez-Rodriguez R, Dalsgaard T, Buus NH, Stankevicius E: Novel approaches to improving endothelium-dependent nitric oxide-mediated vasodilatation. Pharmacol Rep. 2009, 61: 105-115.

    Article  CAS  PubMed  Google Scholar 

  17. Zhang SH, Reddick RL, Burkey B, Maeda N: Diet-induced atherosclerosis in mice heterozygous and homozygous for apolipoprotein E gene disruption. Journal Clin Invest. 1994, 94: 937-945. 10.1172/JCI117460.

    Article  CAS  Google Scholar 

  18. Nakashima Y, Plump AS, Raines EW, Breslow JL, Ross R: ApoE-deficient mice develop lesions of all phases of atherosclerosis throughout the arterial tree. Arterioscler Thromb. 1994, 14: 133-140. 10.1161/01.ATV.14.1.133.

    Article  CAS  PubMed  Google Scholar 

  19. D’Uscio LV, Barton M, Shaw S, Lüscher TF: Chronic ETA receptor blockade prevents endothelial dysfunction of small arteries in apolipoprotein E-deficient mice. Cardiovasc Res. 2002, 53: 487-495. 10.1016/S0008-6363(01)00469-2.

    Article  PubMed  Google Scholar 

  20. Jiang F, Gibson AP, Dusting GJ: Endothelial dysfunction induced by oxidized low-density lipoproteins in isolated mouse aorta: a comparison with apolipoprotein-E deficient mice. Eur J Pharmacol. 2001, 424: 141-149. 10.1016/S0014-2999(01)01140-2.

    Article  CAS  PubMed  Google Scholar 

  21. Dussault S, Maingrette F, Ménard C, Michaud S-É, Haddad P, Groleau J, Turgeon J, Perez G, Rivard A: Sildenafil increases endothelial progenitor cell function and improves ischemia-induced neovascularization in hypercholesterolemic apolipoprotein E – deficient mice. Hypertension. 2009, 54: 1043-1049. 10.1161/HYPERTENSIONAHA.109.139451.

    Article  CAS  PubMed  Google Scholar 

  22. Zadelaar S, Kleemann R, Verschuren L, De Vries-Vander Weij J, van der Hoorn J, Princen HM, Kooistra T: Mouse models for atherosclerosis and pharmaceutical modifiers. Arterioscler Thromb Vasc Biol. 2007, 27: 1706-1721. 10.1161/ATVBAHA.107.142570.

    Article  CAS  PubMed  Google Scholar 

  23. Zhou Q, Liao JK: Pleiotropic effects of statins. - Basic research and clinical perspectives. Circ J. 2010, 74: 818-826. 10.1253/circj.CJ-10-0110.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  24. D’ Uscio LV, Baker TA, Mantilla CB, Smith L, Weiler D, Sieck GC, Katusic ZS: Mechanism of endothelial dysfunction in apolipoprotein E-deficient mice. Arterioscler Thromb Vasc Biol. 2001, 21: 1017-1022. 10.1161/01.ATV.21.6.1017.

    Article  Google Scholar 

  25. Kauser K, da Cunha V, Fitch R, Mallari C, Rubanyi GM: Role of endogenous nitric oxide in progression of atherosclerosis in apolipoprotein E-deficient mice. Am J Physiol Heart Circ Physiol. 2000, 278: H1679-H1685.

    CAS  PubMed  Google Scholar 

  26. Koupparis AJ, Jeremy JY, Muzaffar S, Persad R, Shukla N: Sildenafil inhibits the formation of superoxide and the expression of gp47phox NAD[P]H oxidase induced by the thromboxane A2 mimetic, U46619, in corpus cavernosal smooth muscle cells. BJU Int. 2005, 96: 423-427. 10.1111/j.1464-410X.2005.05643.x.

    Article  CAS  PubMed  Google Scholar 

  27. Vertuani S, Angusti A, Manfredini S: The antioxidants and pro-antioxidants network: an overview. Curr Pharm Des. 2004, 10: 1677-1694. 10.2174/1381612043384655.

    Article  CAS  PubMed  Google Scholar 

  28. Rizzo NO, Maloney E, Pham M, Luttrell I, Wessells H, Tateya S, Daum G, Handa P, Schwartz MW, Kim F: Reduced NO-cGMP signaling contributes to vascular inflammation and insulin resistance induced by high-fat feeding. Arterioscler Thromb Vasc Biol. 2010, 30: 758-765. 10.1161/ATVBAHA.109.199893.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  29. Morihara N, Sumioka I, Ide N, Moriguchi T, Uda N, Kyo E: Aged garlic extract maintains cardiovascular homeostasis in mice and rats. J Nutr. 2006, 136: 777S-781S.

    CAS  PubMed  Google Scholar 

  30. Ohashi M, Runge MS, Faraci FM, Heistad DD: MnSOD deficiency increases endothelial dysfunction in ApoE-deficient mice. Arterioscler Thromb Vasc Biol. 2006, 26: 2331-2336. 10.1161/01.ATV.0000238347.77590.c9.

    Article  CAS  PubMed  Google Scholar 

  31. Cola MS, Gava AL, Meyrelles SS, Vasquez EC: Endothelial dysfunction of resistance vessels in female apolipoprotein E-deficient mice. Lipids Health Dis. 2010, 9: 51-10.1186/1476-511X-9-51.

    Article  PubMed Central  PubMed  Google Scholar 

  32. Villeneuve N, Fortuno A, Sauvage M, Fournier N, Breugnot C, Jacquemin C, Petit C, Gosgnach W, Carpentier N, Vanhoutte P, Vilaine JP: Persistence of the nitric oxide pathway in the aorta of hypercholesterolemic apolipoprotein-E-deficient mice. J Vasc Res. 2003, 40: 87-96. 10.1159/000070705.

    Article  CAS  PubMed  Google Scholar 

  33. Arruda RMP, Peotta VA, Meyrelles SS, Vasquez EC: Evaluation of vascular function in apolipoprotein E knockout mice with angiotensin-dependent renovascular hypertension. Hypertension. 2005, 46: 932-936. 10.1161/01.HYP.0000182154.61862.52.

    Article  CAS  PubMed  Google Scholar 

  34. Custodis F, Baumhäkel M, Schlimmer N, List F, Gensch C, Böhm M, Laufs U: Heart rate reduction by ivabradine reduces oxidative stress, improves endothelial function, and prevents atherosclerosis in apolipoprotein E-deficient mice. Circulation. 2008, 117: 2377-2387. 10.1161/CIRCULATIONAHA.107.746537.

    Article  CAS  PubMed  Google Scholar 

  35. Baumhakel M, Custodis F, Schlimmer N, Laufs U, Böhm M: Improvement of endothelial function of the corpus cavernosum in apolipoprotein E knockout mice treated with irbesartan. J Pharmacol Exp Ther. 2008, 327: 692-698. 10.1124/jpet.108.140533.

    Article  PubMed  Google Scholar 

  36. Chen X, Zhang H, McAfee S, Zhang C: The reciprocal relationship between adiponectin and LOX-1 in the regulation of endothelial dysfunction in ApoE knockout mice. Am J Physiol Heart Circ Physiol. 2010, 299: H605-H612. 10.1152/ajpheart.01096.2009.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  37. Schäfer A, Fraccarollo D, Pförtsch S, Flierl U, Vogt C, Pfrang J, Kobsar A, Renné T, Eigenthaler M, Ertl G, Bauersachs J: Improvement of vascular function by acute and chronic treatment with the PDE-5 inhibitor sildenafil in experimental diabetes mellitus. Br J Pharmacol. 2008, 153: 886-893.

    Article  PubMed Central  PubMed  Google Scholar 

  38. Bivalacqua TJ, Sussan TE, Gebska MA, Strong TD, Berkowitz DE, Biswal S, Burnett AL, Champion HC: Sildenafil inhibits superoxide formation and prevents endothelial dysfunction in a mouse model of secondhand smoke induced erectile dysfunction. J Urol. 2009, 181: 899-906. 10.1016/j.juro.2008.10.062.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  39. Das A, Xi L, Kukreja RC: Phosphodiesterase-5 inhibitor sildenafil preconditions adult cardiac myocytes against necrosis and apoptosis, Essential role of nitric oxide signaling. J Biol Chem. 2005, 280: 12944-12955.

    Article  CAS  PubMed  Google Scholar 

  40. Salloum F, Yin C, Xi L, Kukreja RC: Sildenafil induces delayed preconditioning through inducible nitric oxide synthase-dependent pathway in mouse heart. Circ Res. 2003, 92: 595-597. 10.1161/01.RES.0000066853.09821.98.

    Article  CAS  PubMed  Google Scholar 

  41. Salloum FN, Abbate A, Das A, Houser JE, Mudrick CA, Qureshi IZ, Hoke NN, Roy SK, Brown WR, Prabhakar S, Kukreja RC: Sildenafil (Viagra) attenuates ischemic cardiomyopathy and improves left ventricular function in mice. Am J Physiol Heart Circ Physiol. 2008, 294: H1398-H1406.

    Article  CAS  PubMed  Google Scholar 

  42. Matsumoto T, Miyamori K, Kobayashi T, Kamata K: Apocynin normalizes hyperreactivity to phenylephrine in mesenteric arteries from cholesterol-fed mice by improving endothelium-derived hyperpolarizing factor response. Free Radic Biol Med. 2006, 41: 1289-1303. 10.1016/j.freeradbiomed.2006.07.012.

    Article  CAS  PubMed  Google Scholar 

  43. Porto ML, Lima LC, Pereira TM, Nogueira BV, Tonini CL, Campagnaro BP, Meyrelles SS, Vasquez EC: Mononuclear cell therapy attenuates atherosclerosis in apoE KO mice. Lipids Health Dis. 2011, 10: 155-10.1186/1476-511X-10-155.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  44. Lima LCF, Porto ML, Campagnaro BP, Tonini CL, Nogueira BV, Pereira TM, Vasquez EC, Meyrelles SS: Mononuclear cell therapy reverts cuff-induced thrombosis in apolipoprotein E-deficient mice. Lipids Health Dis. 2012, 11: 96-10.1186/1476-511X-11-96.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  45. Moore KJ, Tabas I: Macrophages in the pathogenesis of atherosclerosis. Cell. 2011, 145: 341-355. 10.1016/j.cell.2011.04.005.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

Download references


ECV and SSM are supported by the National Council for the Development of Science and Technology (CNPq, Ref. 302582/2011-8 and 302535/2009-8 Grants, respectively) and the State Agency for the Development of Science and Technology (FAPES/Universal 012/2011/PROC. 54498465 and FAPES/CNPq/PRONEX Edital 012/2009). The histology was performed at the facilities of the Multiuser Laboratory of Molecular Histology and Immunohistochemistry of the Health Sciences Center of the Federal University of Espirito Santo.

Author information

Authors and Affiliations


Corresponding author

Correspondence to Elisardo C Vasquez.

Additional information

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

CMB and MAL carried out the animal experiments, the data analysis and the statistics and drafted the manuscript. IBSG and ALG contributed to the animal experiments and the data analysis. TMCP and SSM participated designing the study and drafted the manuscript. ECV contributed to the conception, design and supervision of the study, as well as the interpretation of the data. All of the authors have read and approved the final version of the manuscript.

Authors’ original submitted files for images

Rights and permissions

Open Access This article is published under license to BioMed Central Ltd. This is an Open Access article is distributed under the terms of the Creative Commons Attribution License ( ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Reprints and permissions

About this article

Cite this article

Balarini, C.M., Leal, M.A., Gomes, I.B.S. et al. Sildenafil restores endothelial function in the apolipoprotein E knockout mouse. J Transl Med 11, 3 (2013).

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: