Effects of long-and short-term darbepoetin-α treatment on oxidative stress , inflammation and endothelial injury in ApoE knockout mice

Material and methods. In order to study the effect of darbepoetin-α on endothelial injury and atherosclerosis, we used ApoE-/mice as the atherosclerotic mice model. We monitored atherosclerosis and plaque formation histochemically in ApoE knockout mice at early and late stages of atherosclerosis. Darbepoetin-α was injected intraperitoneally at a dose of 0.1 μg/kg to ApoE-/mice. The results of 2 ApoE-/mice groups injected with darbepoetin-α (early and late stages of atherosclerosis) were compared to the results of the corresponding saline injected ApoE-/mice groups and the control (C57BL/6) mice.

The prevalence of cardiovascular diseases (CVD) rises with ageing, and atherosclerosis is the the major underlying cause of CVDs.Atherosclerosis affects large and medium-sized arterial vessels.Interactions of genetic and environmental factors play a major role in atherosclerosis development.Atherosclerosis is defined by vascular inflammation, endothelial dysfunction, accumulation of lipids, cholesterol and cellular debris within the intima of the artery wall.This accumulation results in vascular remodeling, blood flow abnormalities, plaque formation, luminal blockage and decreased distribution of oxygen to target tissues. 1he proposed initial step of atherogenesis is endothelial dysfunction inducing a number of compensatory responses and leading to the alteration of normal vascular homeostatic properties. 2 Atherosclerotic lesions develop as a result of inflammatory stimuli, subsequent release of various cytokines, proliferation of smooth muscle cells, synthesis of connective tissue matrix and accumulation of macrophages and lipids.It is now recognized that inflammation plays a pivotal role in early atherogenesis. 3poE knockout (ApoE −/− ) mice are used most commonly for the study of in vivo experimental models for human atherosclerosis.4 The mouse is the major mammalian model organism for the discovery of genes and mechanisms related to atherosclerosis.Several candidate genes suspected of contributing to the progression of atherosclerosis have been tested through the development and analysis of gene deficient or transgenic mice.6 The ApoE knockout (ApoE −/− ) mice model is presently the most prevalent murine model in cardiovascular research and has given extensive insights into atherosclerosis."These mice develop spontaneous atherosclerotic plaques with many features similar to human lesions and allow follow-up of atherosclerosis process from its earliest stages; moreover it can be used as an ideal model for biomarker discovery".7,8 ApoE −/− mice exhibit chronic hypercholesterolemia and develop early and spontaneous atherosclerotic lesions.9 Organic fluids like serum represent the most convenient samples because of their easy access, providing a means to explore possible markers for atherosclerosis and identify the most appropriate ones for clinical use.Furthermore, such biomarkers have the potential to provide information about different atherosclerosis stages and clinical help from a prognostic point of view.
Darbepoetin-α is a hyperglycosylated erythropoiesis stimulating protein that has an almost 3-fold longer mean terminal half-life than recombinant human erythropoietin, which allows for less frequent dosing.Darbepoetin-α has been defined as tissue protector in the kidney and preserves capillary network.1][12] Darbepoetin-α reduces infarcted myocardium size, weakens cardiomyocyte apoptosis and enhances cardiac function. 13he effects of darbepoetin-α on the biochemical parameters of atherosclerosis and histopathological lesions in apolipoprotein E knockout (ApoE −/− ) mice are not known.In this study, we investigated the effects of darbepoetin-α treatment during atherosclerosis progression and development in ApoE knockout mice fed with a standard diet compared to wild-type C57BL/6 mice having the same genetic profile as the control group.Our aim was to reveal the possible differences in various biochemical parameters regarding lipid profile, inflammation, endothelial injury and oxidative stress in ApoE −/− and control mice groups and to understand the effect of darbepoetin-α on the studied parameters.Histological assessment of the atherosclerotic lesions was carried out in the sections obtained from the aortic arch (arcus aortae) and subjected to immunohistochemical staining from the ApoE −/− mice at 8, 12 and 20 weeks of age, to demonstrate overt signs of atherosclerotic changes, lipid formation and foam cells during atherosclerosis progression.

Laboratory animals
All experimental protocols conducted on mice were performed in mice were maintained in accordance with the Guide of the Animal Care and Use Committee of Akdeniz University and were used in accordance with an animal study proposal approved by the NIAID Animal Care and use Committee (NIH Publication no.86-23, revised 1996) and the ''Principle of Laboratory Animal Care'' (NIH publication no.86 -23, revised 1985).Genetically modified male homozygous ApoE -/-mice were obtained from Taconic Laboratories (Ejby, Denmark) and the control group having the same genetic background except for the ApoE gene (C57BL/6) were obtained from Akdeniz University, Experimental Animal Laboratory, Antalya, Turkey, at 3-4 weeks of age. 14The animals were kept under controlled 12 h light-dark cycles, 23 ± 1°C constant temperature and 50 ± 5% humidity conditions with free access to chow diet and water.

Drug treatment
The ApoE _/_ mice were split into 4 groups (10 animals each) which were injected with darbepoetin-α or saline for a period of 8 or 20 weeks (initial and advanced stages of atherosclerosis, respectively).The darbepoetin-α (Aranesp, Amgen, Thousand Oaks, USA) was dissolved in saline and used in a dose of 0.1 mg/kg/week.The drug solution was freshly prepared on every injection day and administered to the mice intraperitoneally. 15The dose was adjusted according to the body weight of each mouse during the treatment period.

Histological examination of the aorta
For histological examinations to show initial, intermediate and advance atherosclerotic lesion, the ApoE knockout mice were sacrificed at the termination of 8, 12 and 20 weeks of darbepoetin-α treatment.The hearts and aorta of the mice were carefully removed and dissected free from connective tissue from the brachiocephalic trunk to the iliac bifurcation point, washed with cold saline and fixed with 10% neutral buffered formalin, then processed for embedding in paraffin wax. 14For histological assessment of atherosclerosis, the samples were processed in accordance with the usual manner and embedded in paraffin and cut into 4 µm sections.The tissue sections were stained with hematoxylin and eosin.Stained tissue sections were visualized using light microscopy (Leica).

Blood sample collection
Blood samples were collected from the abdominal aorta at the end of the 8 th and 20 th weeks of darbepoetin-α or saline treatment of the ApoE −/− mice and control mice (C57BL/6) under ether anesthesia, and centrifuged at 3000 rpm for 10 min.Each serum sample was separated and stored in aliquots at -80°C with addition of a protease inhibitor cocktail (Catalog no.#S8820, Sigma-Aldrich, St. Louis, USA) to avoid protein degradation during the preparation of the samples. 16

Triglyceride assay
Triglyceride levels were determined using the enzymatic colorimetric method of the Roche Diagnostics assay kit (Catalog no.#11730711, Indianapolis, Indiana, USA) in the Roche Hitachi Modular P800-ISE autoanalyzer.

Cholesterol assay
Cholesterol levels were determined using the enzymatic colorimetric Roche Diagnostics assay kit (Catalog no.#11491458, Indianapolis, USA) in the Roche Hitachi Modular P800-ISE autoanalyzer.

Protein carbonyl colorimetric assay
Protein-bound carbonyls were measured using a protein carbonyl assay kit (Catalog no.#1005020, Cayman Chemical, Ann Arbor, USA).The utilized method was based on the covalent reaction of the carbonylated protein side chain with 2,4-dinitrophenylhydrazine (DNPH) and detection of the produced protein hydrazone at an absorbance of 370 nm.The results were calculated using the extinction coefficient of 22 mM -1 cm -1 for aliphatic hydrazones and expressed as nmol/mg protein.

Interleukin-6 (IL-6) assay
A mouse IL-6 ELISA kit (Catalog no.#550950, BD OptEIA, BD Biosciences, USA) was used for the quantitative detection of serum IL-6.An IL-6 coated plate specific for monoclonal antibody was utilized.Each IL-6 present in the serum binds to the immobilized antibody.Streptavidin horseradish peroxidise (HRP) conjugate was mixed with biotinylated anti-mouse IL-6 antibody, producing an antibody-antigen-antibody "sandwich".The plate was washed and TMB solution was added, which produced color in direct proportion to the amount of IL-6 present in the original sample, and absorbances measured at 450 nm.

Histamine assay
Histamine levels were determined using an assay kit (Catalog no.#A05890, SPI BIO, Massy, France).This method depends on the competition between unmarked derivatized histamine and acetylcholinesterase linked to a histamine tracer for a limited number of mouse anti-histamine antibody sites.

Mouse C-reactive protein (CRP) assay
CRP levels were measured using an assay kit (Catalog no.#41-CRPMS-E01, Alpco Diagnostics, Salem, USA).The CRP present in serum reacts with the anti-CRP antibodies coated to the surface of the wells.After washing, anti-CRP antibodies conjugated with horseradish peroxidase were added, forming complexes with the previously bound CRP.Enzyme bound to the immunosorbent was assayed by the addition of a chromogenic substrate.The bound enzyme quantity depends on the concentration of CRP in the serum.

E-selectin assay
E-selectin levels were evaluated with a quantitative sandwich enzyme immunoassay kit (Catalog no.#MES00, R&D Systems, Minneapolis, USA).A microplate was endothelial space, formation of a lipid core, thickening of the intima-media and narrowing of the lumen. 14Changes in the histopathological grade of atherosclerosis was significantly different among the groups.No atherosclerotic lesions were observed in the control mice.The median value of the aorta lumen was highest in the aorta of the control mice and lowest in the 20 week-old ApoE -/- mice.The first atherosclerotic lesions were developed and characterized in the 8-week-old ApoE -/-mice group.Lipid laden macrophage formation was observed in the 12-week-old ApoE -/-mice group.Extracellular lipid formation and foam cells were seen in the 20-week-old ApoE -/-mice.

Biochemical analyses Triglyceride levels
Triglyceride levels in the darbepoetin-α-treated and non-treated atherosclerotic ApoE −/− mice groups at the initial and advanced stages of atherosclerosis were significantly increased compared to the control (C57BL/6) group (p < 0.001) (Fig. 2).Darbepoetin-α treatment caused a significant increase in triglyceride levels compared to the non-treated ApoE knockout mice group at the initial atherosclerotic stage (p < 0.05).In contrast, triglyceride levels were not significantly affected by the darbepoetin-α treatment in the advanced stage groups.The triglyceride levels of the darbepoetin-α-treated and non-treated ApoE knockout mice groups at the advanced atherosclerotic stage were significantly higher compared to the darbepoetin-α-treated and non-treated ApoE knockout mice groups at the initial atherosclerotic stage (p < 0.05).

Cholesterol level
Serum cholesterol levels of the darbepoetin-α-treated, and non-treated ApoE knockout mice groups at the initial and advanced stages of atherosclerosis were significantly increased compared to the C57BL/6 mice (p < 0.001), as shown in Fig. 3. Cholesterol levels were not significantly affected by darbepoetin-α treatment in ApoE knockout mice groups at the initial and advanced atherosclerotic stages (p > 0.05).Darbepoetin-α-treated, and non-treated ApoE -/-mice groups at the advanced stage had significantly higher cholesterol levels compared to the darbepoetin-α-treated, and non-treated ApoE knockout mice groups at the initial stage (p < 0.05).

Protein carbonyl levels
Protein carbonyl levels were markedly higher in the 4 ApoE -/-mice groups compared to the C57BL/6 mice group (p < 0.001), as shown in Fig. 4. Darbepoetin-α treatment decreased the protein carbonyl level in the ApoE -/- pre-coated with a monoclonal antibody specific to mouse E-selectin.An immobilized antibody was utilized for binding of any mouse E-selectin present in the serum.After the washing steps, E-selectin specific enzyme-linked monoclonal antibody was added to the wells.Following a wash, a substrate solution was added to the wells.The enzyme reaction yielded a blue product that turned yellow when the Stop Solution was added.The intensity of the color was proportional to the amount of mouse E-selectin bound in the first step.The minimum detectable dose of mouse E-selectin was 2.8-4.7 pg/mL.The mean minimum detectable dose was 3.8 pg/mL.

Statistical analysis
Statistical analyses were carried out using SPSS packed program v. 17 (IBM, Chicago, USA).Data was expressed as mean ± standard deviation (SD).Normal distribution and differences between variances were determined using Kolmogorov-Smirnov and Levene tests, respectively.For comparisons between the 2 groups, the Student's t-test and Mann-Whitney U-test were used as appropriate.For subgroup analyses, the Kruskal-Wallis test was used to determine significant differences.The Mann-Whitney U-test was used to determine differences between the groups if a significant difference was found in the Kruskal-Wallis test.P-value < 0.05 was considered statistically significant.

Animal data
At the end of the study, the body weights were not significantly different among the groups.We did not observe any differences in animal behavior or activity in ApoE -/-mice compared to the controls.

Histopathological findings
Atherosclerotic lesions of the aortic arch are shown for the control (C57BL/6) mice (Fig. 1a), 8-week-old ApoE -/- mice (Fig. 1b), 12-week-old ApoE -/-mice (Fig. 1c) and twenty-week-old ApoE -/-mice (Fig. 1d).The ApoE -/-mice developed early atherosclerotic lesions in the aortic root, characterized by accumulation of foam cells in the sub-mice groups at both the initial and advanced atherosclerotic stages, but this diminution was significant only in the ApoE -/-mice group at the advanced atherosclerotic stage (p < 0.05).

8-isoprostane level
8-Isoprostane levels were significantly higher in 4 ApoE -/- mice groups compared to the C57BL/6 mice group (p < 0.001), as shown in Fig. 5. Darbepoetin-α treatment significantly decreased 8-Isoprostane levels in the ApoE -/- mice groups at the initial and advanced atherosclerotic stages compared to the corresponding non-treated ApoE -/- mice groups at the initial and advanced atherosclerotic stages (p < 0.01 and p < 0.05 respectively).

Interleukin-6 (IL-6) level
IL-6 levels were significantly enhanced in 4 ApoE -/- mice groups compared to the C57BL/6 mice group (p < 0.001), as shown in Fig. 6.The highest level was found in the non-treated ApoE -/-mice group at the advanced atherosclerotic stage.There was no marked difference in IL-6 levels among 4 ApoE -/-mice groups (p > 0.05).Darbepoetin-α treatment decreased IL-6 levels in ApoE -/- mice groups at the initial and advanced atherosclerotic stages compared to the corresponding non-treated ApoE knockout mice groups at the initial and advanced atherosclerotic stages, but these decreases were not significant (p > 0.05).

Histamine level
Histamine levels were significantly higher in 4 ApoE -/- mice groups compared to the C57BL/6 mice group (p < 0.05), as shown in Fig. 7.The highest level was found in the non-treated ApoE knockout mice group at the advanced atherosclerotic stage compared to the other 3 ApoE knockout mice groups, although these differences among 4 ApoE -/-mice groups were not significant (p > 0.05).Darbepoetin-α treatment decreased histamine levels in ApoE -/-mice groups at the initial and advanced atherosclerotic stages compared to the corresponding non-treated ApoE -/-mice groups at the initial and advanced atherosclerotic stages, but these decreases were not significant (p > 0.05).

C-reactive protein (CRP) levels
CRP levels were significantly higher in the four ApoE -/- mice groups compared to the C57BL/6 mice group (p < 0.01), as shown in Fig. 8. CRP level was significantly higher in the non-treated ApoE knockout mice group at the advanced stage compared to the non-treated ApoE knockout mice group at the initial stage (p < 0.01).Darbepoetin-α treatment decreased CRP levels in ApoE knockout mice groups at the initial and advanced atherosclerotic stages compared to the corresponding non-treated ApoE knockout mice groups at the initial and advanced atherosclerotic stages, but these decreases were not significant (p > 0.05).

E-selectin level
The levels of E-selectin were markedly higher in 4 ApoE -/- mice groups compared to the C57BL/6 mice group (p < 0.01), as shown in Fig. 9.There was a non-significant increase in E-selectin level in the non-treated ApoE knockout mice group at the advanced stage compared to the non-treated ApoE knockout mice group at the initial stage (p > 0.05).Darbepoetin-α treatment nonsignificantly decreased the E-selectin level in ApoE -/-mice groups at the advanced atherosclerotic stage compared to the non-treated ApoE knockout mice group at the advance atherosclerotic stage.

ICAM-1 (CD54) level
Soluble intercellular cell adhesion molecule levels were significantly higher in 4 ApoE -/-mice groups compared to the C57BL/6 mice group (p < 0.01), as shown in Fig. 10.There was a marked increase in ICAM-1 level in the non-treated ApoE knockout mice group at the advanced stage compared to the non-treated ApoE -/-mice group at the initial stage (p < 0.05).Darbepoetin-α treatment decreased ICAM-1 level in ApoE -/-mice groups at the initial and advanced atherosclerotic stages compared to the corresponding non-treated ApoE knockout mice groups at the initial and advanced atherosclerotic stages, but these decreases were not significant (p > 0.05).

Discussion
Our study explained the differences in various biochemical parameters regarding lipid profile, inflammation, endothelial injury and oxidative stress in ApoE −/− mice receiving darbepoetin-α or saline and the effects of darbepoetin-α on the studied parameters.C57BL/6 mice, having the same genetic background except the ApoE gene served as the control group. 14Histological assessment of the atherosclerotic lesions to demonstrate overt signs of atherosclerotic changes, lipid accumulation, foam cell and plaque formation during atherosclerosis progression was carried out in the sections obtained from the aortic arch (arcus aortae) and subjected to immunohistochemical staining from the ApoE −/− mice at 8, 12 and 20 weeks of age.From the initial to advanced stage of atherosclerosis, the median value of the aorta lumen was Fig. 7. Serum histamine level of the control (C57BL/6), IA, IAD, AA and AAD groups.Data is expressed as mean ± standard deviation (SD) Fig. 8. CRP level of the control (C57BL/6), IA, IAD, AA and AAD groups.Data is expressed as mean ± standard deviation (SD) Fig. 9. Serum E-selectin level of the control (C57BL/6), IA, IAD, AA and AAD groups.Data is expressed as mean ± standard deviation (SD) Fig. 10.sICAM-1 level of the control (C57BL/6), IA, IAD, AA and AAD groups.Data is expressed as mean ± standard deviation (SD) *p < 0.05, darbepoetin-α-treated and non-treated ApoE -/-mice groups at the initial and advanced atherosclerotic stages vs the control group.*p < 0.01, darbepoetin-α-treated and non-treated ApoE -/-mice groups at the initial and advanced atherosclerotic stages vs the control group; **p < 0.01, Non-treated ApoE -/-mice group at the advanced stage vs nontreated ApoE -/-mice group at the initial stage.* p < 0.01, darbepoetin-α-treated and non-treated ApoE -/-mice groups at the initial and advanced atherosclerotic stages vs the control group.*p < 0.01, darbepoetin-α-treated and non-treated ApoE -/-mice groups at the initial and advanced atherosclerotic stages vs the control group; **p < 0.05, non-treated ApoE -/-mice group at the advanced stage vs nontreated ApoE -/-mice group at the initial stage.
diminished, indicating narrowing of the lumen gradually.Atherosclerotic lesion development was first seen at the age of 8 weeks in the ApoE -/-mice.At the 20 th week of age, ApoE knockout mice developed foam cells in the aortic arch.Our observations are in accordance with the studies of Jawien et al., who reported that the ApoE knockout mice model is the only one that develops large-scale accelerated atherosclerotic lesions. 17polipoprotein E gene mutations increase cholesterol and triglyceride levels leading to accelerated atherosclerosis. 18In fact, the ApoE -/-mice used in our study had hypercholesterolemia and hypertriglyceridemia and developed accelerated atherosclerosis. 9The increases in triglyceride and cholesterol levels were in parallel with the stages of atherosclerosis in the ApoE knockout mice.We evaluated the short and long term responses of darbepoetin-α on the characteristics of atherosclerosis in ApoE −/− mice.Darbepoetin-α treatment significantly increased triglyceride and cholesterol levels compared to the saline-treated ApoE −/− mice groups at the initial and advanced stages of atherosclerosis.
We determined oxidative stress parameters such as 8-isoprostane as a lipid peroxidation index and protein carbonyl as a protein oxidation index in the control and ApoE -/-mice groups.8-isoprostane and protein carbonyl levels were significantly increased in the ApoE -/-mice groups compared to the C57BL/6 mice group, indicating an increase in oxidative stress during atherosclerosis development.Darbepoetin-α treatment significantly lowered enhanced oxidative stress marker levels in the ApoE knockout mice in comparison to the saline-injected ApoE -/-mice.The decreases in 8-isoprostane levels by darbepoetin-α treatment were significant in the ApoE -/- mice groups at the initial and advanced atherosclerotic stages, but the decreases in protein carbonyl levels were significant in the ApoE -/-mice group only at the advanced atherosclerotic stage compared to the corresponding non-treated ApoE knockout mice groups at the initial and advanced atherosclerotic stages.Our findings of diminishing oxidative stress markers in ApoE -/-mice receiving darbepoetin-α at the initial or advanced atherosclerotic stages, when compared to the corresponding non-treated ApoE -/-mice groups, indicate the antioxidative effects of darbepoetin-α.
Although long term darbepoetin-α treatment reduced oxidative stress parameters in ApoE knockout mice, and it did not change the markers of lipid profile (compared to the untreated, advanced atherosclerotic group) and endothelial injury, similar to the reports by Monostori et al. and Parissis et al. 19,20 Increased plasma protein carbonyls in patients with hypercholesterolemia have been reported, indicating that the plasma protein carbonyl is a sensitive index of oxidative stress. 20,21ur findings showed that IL-6 and CRP levels, well known inflammation markers, were raised in parallel with the stage of atherosclerosis in the non-darbepoetin-α-treated ApoE knockout mice group. 22Darbepoetin-α treatment decreased these inflammation markers in the ApoE -/-mice both at the advanced and initial atherosclerosis stages.Related to our findings, Wilund et al. published that IL-6 and CRP were enhanced in ApoE knockout mice compared to ApoE +/+ mice. 23In accordance with our findings, Arend et al. reported a significant decrease in CRP expression in darbepoetin-α-treated ApoE -/-mice. 24In a study performed by Huang et al. it was reported that basal serum IL-6 levels of ApoE knockout mice were akin to those in C57BL mice. 25istamine and its receptors are formed in atherosclerotic lesions, and their signaling and following proinflammatory and proatherogenic gene expression are involved in the progress of atherogenesis.Wang et al. showed that histamine was associated with the development of atherosclerosis in ApoE knockout mice by regulating the gene expression of inflammatory modulators independent of serum cholesterol levels.In addition, histamine participates in hyperlipidemia-induced atherosclerosis. 26Huang et al. reported that serum histamine in ApoE knockout mice was markedly increased compared to the level in control (C57BL) mice. 27Serum histamine, CRP and IL-6 levels were shown to be increased in acute coronary syndrome and stable coronary artery disease. 28In our study, darbepoetin-α treatment decreased histamine levels in ApoE -/-mice groups at the initial and advanced atherosclerotic stages compared to the corresponding non-treated ApoE -/-mice, although these decreases were not statistically significant.E-selectin, IL-6 and histamine levels were not affected significantly by darbepoetin-α administration between ApoE -/-mice groups at both initial and advanced stages.
During the progression of atherosclerosis from the initial stage to the advanced stage, elevated E-selectin and ICAM-1 levels were observed in the non-treated ApoE -/- mice.We found increased E-selectin and ICAM-1 levels as indicators of endothelial injury in the non-treated ApoE knockout mice group at the advanced stage of atherosclerosis compared to the non-treated ApoE knockout mice group at the initial stage, but only the increase in ICAM-1 level was significant.Elevated ICAM-1 levels in ApoE -/-mice were also reported by Liyama et al. 29 Rubio-Guerra et al. found a significant correlation between maximal carotid artery intimal-medial thickness and ICAM-1 levels, whereas correlation was not observed with E-selectin in patients with diabetes-associated atherosclerosis. 30In our study, darbepoetin treatment led to a non-significant decrease in ICAM-1 level in the ApoE -/- mice group compared to the corresponding non-darbepoetin-α treated ApoE -/-mice at both initial and advanced atherosclerosis stages.Arend et al. reported that ICAM protein expression in the aorta did not differ markedly between the nephrectomized or darbepoetin-α-treated groups, neither in the intramyocardial arteries and aortic plaque nor in the aortic endothelium of ApoE -/-mice. 24ndothelial function is impaired in the earlier stages of atherogenesis and is strongly correlated with several risk factors.Endothelial dysfunction predisposes to longterm atherosclerotic lesions and has been proposed as an important diagnostic and prognostic factor.The production of oxidative reactive radicals induces dysfunction of the endothelium, the first step of atherogenesis.In such studies, the specificity of the preferred parameters for the definition of atherosclerosis needs to be tested in specimens."The biochemical analyses revealed that several parameters in serum might be involved in the development and/or progression of atherosclerosis in ApoE -/-mice and these parameters could be affected by the darbepoetin-α treatment". 16n our study, we compared the selected markers of lipid profile, inflammation, endothelial injury and oxidative stress in atherosclerotic ApoE knockout mice treated or non-treated with darbepoetin-α.We revealed that long term darbepoetin-α treatment reduced oxidative stress and inflammation in ApoE -/-mice, but did not influence lipid profile (compared to the untreated advanced atherosclerotic group) and endothelial injury significantly.Our study demonstrated the efficacy of darbepoetin-α in reducing CRP and protein carbonyl levels in ApoE knockout mice during atherosclerosis progression.We conclude that darbepoetin-α is efficient in reducing inflammation and oxidative stress, which are potent inducers of atherosclerosis in ApoE -/-mice.