Erythropoietin increases expression and function of vascular CuZn-superoxide dismutase

Abstract

Previous studies have shown that treatment with erythropoietin (EPO) exerts vascular protective effects. The exact mechanisms responsible for these effects are not completely understood. In the present study we hypothesized that EPO stimulates expression and activity of CuZn-superoxide dismutase (SOD1) thus protecting vascular tissue from oxidative stress induced by excessive concentrations of superoxide anions. EPO treatment of wild type mice for two weeks (1000 U/kg, s.c., biweekly) significantly increased aortic expression of SOD1. This effect resulted in significant reduction of superoxide anion concentrations in aorta of treated mice. The ability of EPO to reduce vascular production of superoxide anions was abolished in SOD1-deficient mice. In mouse model of wire-induced injury of common carotid artery, treatment of wild-type mice with EPO prevented pathological remodeling whereas the vascular effect of EPO was absent in SOD1-deficient mice. Our findings demonstrate that treatment with EPO increases vascular expression of SOD1. This effect appears to be an important molecular mechanism underlying vascular protection by EPO.

Introduction

Under physiological conditions, endothelium-derived nitric oxide (NO) exerts vascular protective effects as it dilates the vasculature, prevents adhesion of circulating blood cells and inhibits vascular smooth muscle cell (VSMC) proliferation.¹ The formation of superoxide anions, a major chemical inactivator of NO, is kept under tight control by endogenous superoxide dismutase (SOD) enzymes, which catalyze the conversion of superoxide anions to H2O2 and molecular oxygen.² Three SOD isoforms are known to exist: constitutive copper- and zinc-containing SOD (SOD1), manganese-containing SOD (SOD2), and extracellular-SOD (SOD3).² In most tissues, SOD1 is expressed in the cytosol, while in contrast, SOD2 is exclusively located in mitochondria. The third and the most recently discovered SOD3 is also a copper- and zinc-containing enzyme that following secretion by VSMCs becomes bound to the endothelial surface.³ The SOD1 isoform accounts for 50-80% of total SOD activity and is the predominant form of SOD in blood vessels.^4-6 Most importantly, SOD1 protects intracellular NO bioavailability in endothelial cells by limiting increase in superoxide anions thereby preserving normal intracellular concentration of NO.^7,6

Erythropoietin (EPO) has been recognized as a “tissue protective” cytokine.⁸ In the vasculature, protective effects of EPO are dependent on activation of endothelial nitric oxide synthase (eNOS) and biosynthesis of tetrahydrobiopterin.^9-12 Furthermore, we have previously shown that endothelium-dependent relaxations were normalized in injured carotid arteries of wild-type mice treated with EPO.¹¹ However, the local concentration of NO in the arterial wall is not only dependent on enzymatic activity of eNOS, but is also critically affected by SOD activity and the concentration of superoxide anions. Indeed, high local concentrations of superoxide anions are considered a major mechanism of endothelial dysfunction following vascular injury.^1,13 In the present study, we tested the hypothesis that EPO exerts antioxidant effect in arterial wall by increasing expression and activity of SOD1. To test this hypothesis, we employed SOD1-deficient (SOD1^-/-) mice and determined the effect of EPO on superoxide anion production in SOD1^-/- mice arteries.

Materials and Methods

Experimental Animals And Model of Vascular Injury

Male SOD1^-/- mice (B6;129S7-Sod1^tm1Leb/J) and their wild-type littermates were obtained from Jackson Laboratory (Bar Harbor, ME) and were maintained on standard chow with free access to drinking water. Housing facilities and all experimental protocols were approved by the Institutional Animal Care and Use Committee of the Mayo Clinic and complied with the National Institute of Health Guide for the Care and Use of Laboratory Animals. Wire-induced vascular injury was performed in the left common carotid artery as described previously.¹¹ Mice were randomly distributed to an injury group (PBS, Gibco) and an injury + EPO group (recombinant human EPO alpha 1000 U/kg body weight, biweekly, s.c.; Amgen Thousand Oaks, CA). The dosage of EPO was selected based on previous studies.^14,11 After 14 days treatment, the animal were euthanized (pentobarbital, 60 mg/kg body weight, i.p.).

Systolic Blood Pressure

Systolic blood pressure (SBP) was recorded in quiescent mice by a tail-cuff method (Harvard Apparatus Ltd., Kent, England) before surgery and on fourteenth day of treatment.¹¹

Blood Cell Count

Blood cell counts were performed with ABAXIS VetScan HM2™ Hematology System (Union City, CA) as described.¹¹

Morphological Analysis of Carotid Arteries

Morphological analyses were performed on perfused and fixed vessels in buffered formalin (10%). Each artery was embedded in paraffin, and cross sections were continuously cut every 100 μm from one edge to the other edge of carotid artery (8 sections). Corresponding sections of contra lateral artery were used as control. Each section was mounted on slides and subjected to standard Verhoeff-Van Gieson staining.¹⁵ Adobe Photoshop software 6.0 was used to analyze medial cross sectional area (CSA).

Western Blot Analysis

Aortas and lungs were excised and homogenized in lysis buffer. Equal amounts of protein (50 μg) were separated by SDS-PAGE and transferred to nitrocellulose membrane (Amersham), after which the membranes were probed using primary antibodies against SOD1, SOD2, SOD3 (StressGen), Akt1, pAkt1(Ser⁴⁷³), 3-nitrotyrosine (Upstate), and catalase (Sigma). As a loading control, blots were rehybridized with anti-β-actin (Sigma).¹⁶

Detection of superoxide anions

Intracellular superoxide anions were quantified using HPLC/fluorescence assay that employs dihydroethidium as a probe.¹⁷ A stable fluorescent product 2-hydroxyethidium is formed from the reaction between dihydroethidium and superoxide anions. Aortas were opened longitudinally and incubated in Krebs-HEPES buffer containing 50 μM dihydroethidium (Molecular Probes) at 37°C for 15 minutes. The samples were washed to remove free probe and incubated in Krebs-HEPES buffer for one additional hour. The arteries were then homogenized in 4°C cold methanol and centrifuged at 12,000 rpm. The supernatant was analyzed by HPLC/fluorescence (Beckman Coulter) in 37% acetonitrile in 0.1% trifluoroacetic acid aqueous solution. Data were quantified using 2-hydroxyethidium standard from the reaction between dihydroethidium and Fremy’s salt as described¹⁸ and normalized against tissue protein levels.

Measurement of Hydrogen Peroxide

An Amplex® Red hydrogen peroxide (H₂O₂)/peroxidase Assay kit (Invitrogen) was used to perform the measurements of H₂O₂ release from mouse aorta as previously described.¹⁹

Calculations and Statistical Analysis

All results are expressed as means ± SEM and “n” indicates the number of animals from which tissues were harvested. Single values were compared by one-way ANOVA with Bonferroni’s correction for multiple comparisons. When simple comparisons were made between two groups, where appropriate an unpaired Student’s t-test was used. A value of P<0.05 was considered significant.

Results

SOD1 Protein Expression

Treatment with EPO for 14 days increased protein expression of SOD1 in the aorta of wild-type mice (Figure 1A). In tandem, basal superoxide anion levels decreased (Figure 1B). In contrast, treatment with EPO did not increase expression of SOD2 and SOD3 isoform proteins in wild-type mice (Figure 2). Likewise, EPO had no effect on SOD2 and SOD3 protein expressions in SOD1^-/- mice (Figure 2).

Figure 1.

A. Representative Western blot analysis for expression of SOD1 protein in wild-type mouse aortas after 14 days treatment with EPO (upper panel). The bar graph indicates the results of the relative densitometry as compared with β-actin (bottom panel). B: Quantitative analysis of production of superoxide anions, as detected by 2-hydroxyethidium, in the aorta of wild-type littermates. Data are shown as mean ± SEM (n=6-8). * P<0.05 vs. control wild-type mice (unpaired t-test).

Figure 2.

Effect of 2 weeks treatment with EPO on protein expressions of MnSOD and EC-SOD in the aorta of wild-type and SOD1^-/- mice. A: Representative Western blot analysis for expressions of MnSOD and EC-SOD proteins. The bar graph indicates the results of the relative densitometry as compared with β-actin (B +C). Data are shown as means ± SEM (n=3-5).

Characteristics of SOD1^-/- Mice

Red blood cell number, hematocrit and hemoglobin were significantly reduced in SOD1^-/- mice when compared to wild-type littermates (P<0.05; Table 1). The white blood cell count was unaltered (Table 1). Treatment with EPO for 14 days selectively increased red blood cell profile to a similar degree in both wild-type and SOD1^-/- mice (P<0.05; Table 1).

Table 1.

Effect of 2 weeks treatment with EPO on blood cells profile and systolic blood pressure in wild-type littermates and SOD1^-/- mice.

Parameters	WT	WT + EPO	SOD1-/-	SOD1-/- + EPO
Red blood cells (106/mm3)	9.9±0.3	12.8±0.1 *	8.3±0.1 *	10.6±0.3 *†
Hematocrit (%)	45.8±1.1	60.8±1.2 *	42.2±0.5 *	54.9±0.6 *†
Hemoglobin (g/dL)	15.3±0.4	20.8±0.2 *	13.6±0.2 *	18.1±0.2 *†
White blood cells (103/mm3)	7.1±0.7	9.2±0.6	5.2±0.3	6.9±0.4
Lymphocytes (103/mm3)	5.6±0.5	6.8±0.5	4.1±0.2	5.1±0.3
Monocytes (103/mm3)	0.4±0.1	0.4±0.2	0.1±0.1	0.2±0.1
Granulocytes (103/mm3)	1.1±0.3	2.0±0.2	1.0±0.1	1.7±0.2
Platelets (103/mm3)	682±52	715±22	681±38	768±70

WT indicates wild-type littermates; EPO, erythropoietin. Data are means ± SEM (n=6-8).

^*P<0.05 vs. WT littermates

^†P<0.05 vs. SOD1^-/- mice (ANOVA + Bonferroni’s).

In agreement with previous report⁶, SBP was significantly lower in SOD1^-/- mice as compared with wild-type littermates (P<0.05; Table 2). Administration of EPO for 14 days did not affect SBP in wild-type or SOD1^-/- mice (Table 2).

Table 2.

Systolic blood pressure of wild-type and SOD1^-/- mice and those treated with EPO for 14 days.

Week	WT	WT + EPO	SOD1-/-	SOD1-/- + EPO
0	115±2	118±1	106±2 *	106±2 *
2	119±1	117±1	105±2 *	109±1 *

WT, wild-type littermates; EPO, erythropoietin. Data are mmHg expressed as means ± SEM of 5-7 mice.

^*P<0.05 vs. WT mice (ANOVA + Bonferroni’s).

Effects of EPO on Superoxide Anions Production

Genetic deletion of SOD1 increased MnTBAP-inhibitable superoxide anions levels in SOD1^-/- mice aortas (P<0.05; Figure 3A). Superoxide anion production was quantitatively the same in comparison of in-vitro incubation of wild-type mouse aorta with the Cu²⁺-chelator diethyldithiocarbamic acid, an agent that inhibits both SOD1 and SOD3, indicating that intracellular superoxide anion regulation is dependent on activity of SOD1 (data not shown). Fourteen days treatment with EPO did not prevent elevation of superoxide anions levels in SOD1^-/- mice (Figure 3A).

Figure 3.

A. Quantitative analysis of production of superoxide anions, as detected by 2-hydroxyethidium, in mouse aorta. Superoxide anions levels in wild-type and SOD1^-/- mice in the presence and absence of cell-permeable SOD mimetic Mn(III) tetra(4-benzoic acid) porphyrin chloride (MnTBAP; 10 μM). Results are expressed in nmol/mg protein and are means ± SEM (n=4-7). * P<0.05 vs. control wild-type mice; † P<0.05 vs. without MnTBAP (ANOVA with Bonferroni’s). B: Representative Western blot analysis for expression of 3-nitrotyrosine positive protein in the lung of wild-type and SOD1^-/- mice. β-actin was shown as loading control (bottom panel). C: The bar graph indicates the results of the optical densitometry (O.D.) as quantified by UN-SCAN-IT® (Silk Scientific Inc., Utah). Data are shown as mean ± SEM (n=4-6). * P<0.05 vs. wild-type mice (ANOVA with Bonferroni’s).

Western blot analysis with 3-nitrotyrosine antibody revealed a significant increase in 3-nitrotyrosine abundance in the lungs of SOD1^-/- mice (P<0.05; Figure 3B). EPO treatment for 2 weeks did not affect increased levels of 3-nitrotyrosine positive proteins in SOD1^-/- mice (Figure 3C).

Morphology of Injured Carotid Artery

Under basal conditions, medial thickness was significantly smaller in SOD1^-/- mice as compared to their wild-type littermates (P<0.05; Figure 4). Fourteen days after carotid artery mechanical injury, medial CSA significantly increased in both wild-type and SOD1^-/- mice as compared to corresponding uninjured arteries (P<0.05; Figure 4). Surprisingly, the injury induced increase in medial thickness was significantly smaller in SOD1^-/- mice as compared to wild-type littermates (P<0.05; Figure 4E). Treatment with EPO for 14 days significantly decreased medial CSA of injured carotid arteries in wild-type mice, while EPO treatment did not significantly affect wall thickness of injured carotid arteries in SOD1^-/- mice (Figure 5).

Figure 4.

Morphological studies of carotid arteries of wild-type littermates and SOD1^-/- mice fourteen days after injury. Carotid arteries were stained with standard Verhoeff van-Giessen. Representative photomicrographs of the uninjured carotid arteries of wild-type mice (A) and SOD1^-/- mice (B) carotid arteries, and injured carotid arteries of wild-type mice (C) and SOD1^-/- mice (D). The media is demarcated by internal elastic lamina (open arrow) and external elastic lamina (black arrow). Original magnification ×200. Size bar = 50 μm. E: Quantification of medial CSA in carotid arteries of wild-type littermates and SOD1^-/- mice. Data are shown as means ± SEM (n=11-15). * P<0.05 vs. uninjured carotid arteries of wild-type mice; † P<0.05 vs. uninjured carotid arteries of SOD1^-/- mice; # P<0.05 vs. wild-type mice after carotid injury (ANOVA with Bonferroni’s).

Figure 5.

Effect of EPO on morphological changes of injured carotid arteries in wild-type and SOD1^-/- mice. Results are means ± SEM (n=7-15) and expressed as percentile changes from uninjured carotid arteries. * P<0.05 vs. PBS treated wild-type mice; n.s. not statistically different (ANOVA with Bonferroni’s).

Akt Activation in SOD1^-/- Mice

Protein kinase B (PKB or Akt) is a critical component of a major signaling pathways involved in cellular proliferation, migration and survival – events that contribute to vascular hyperplasia and restenosis.²⁰ In the current study, Western blot analysis showed a significant decrease in Akt1 phosphorylation at Ser⁴⁷³ in SOD1^-/- mice aortas as compared to wild-type littermates (P<0.05; Figure 6). In these wild-type littermates, treatment with EPO for 2 weeks significantly increased Akt1 phosphorylation at Ser⁴⁷³ (P<0.05; Figure 6). In contrast, EPO failed to activate Akt phosphorylation in SOD1^-/- mice (P<0.05 vs. EPO-treated wild-type littermates; Figure 6). Akt1 protein (total) remained constant and did not differ between wild-type and SOD1^-/- mice.

Figure 6.

Effect of 2 weeks treatment with EPO on protein expression of Ser⁴⁷³-phosphorylated Akt1 in the aorta of wild-type and SOD1^-/- mice. A: Representative Western blot analysis for expression of Ser⁴⁷³-pAkt1 and Akt1 proteins. B: The bar graph indicates the results of the relative densitometry compared with Akt1. Data are shown as means ± SEM (n=5-8 independent experiments). * P<0.05 vs. wild-type mice; † P<0.05 vs. EPO-treated wild-type mice (ANOVA with Bonferroni’s).

H₂O₂ Release and Catalase Expression in SOD1^-/- Mice

H₂O₂ release from the aorta was significantly decreased in SOD1^-/- mice as compared to wild-type mice (P<0.05; Figure 7A). Furthermore, protein expression of catalase was significantly increased in the aorta of SOD1^-/- mice (P<0.05; Figure 7B and 7C).

Figure 7.

A. Quantitative analysis of H₂O₂ release from the aorta of wild-type and SOD1^-/- mice. Data are shown as mean ± SEM (n=5-6) and are expressed in nmol/mg protein per hour. 7B: Representative Western Blot analysis for expression of catalase in the aorta of wild-type and SOD1^-/- mice. C: Bar graph indicates the results of the relative densitometry normalized to β-actin levels. Results are means ± SEM (n=3). * P<0.05 vs. wild-type mice littermates (unpaired t-test).

Discussion

In the present study, we report several novel findings. First, treatment with EPO for two weeks increased protein expression of SOD1 and decreased superoxide anions concentrations in the aorta of wild-type mice. Second, EPO did not affect increased superoxide anions levels in SOD1^-/- mice. Third, EPO treatment significantly prevented aberrant remodeling of injured carotid arteries in wild-type mice but not in SOD1^-/- mice. Thus, our results suggest that augmenting expression and activity of SOD1 by EPO is an important mechanism responsible for prevention of oxidative stress.

EPO is widely used in clinical practice to correct anemia. Most recently, several studies have shown that EPO has vasoprotective effects – an action that is independent of erythropoiesis.^9-11 Impaired NO-mediated endothelium-dependent relaxations and increased medial thickness after injury were normalized in wild-type mice treated with EPO.¹¹ Interestingly, our present study showed that treatment with EPO selectively increased vascular SOD1 protein expression and decreased superoxide anion levels in wild-type mice but did not affect superoxide anion in SOD1^-/- mice. We regard this observation as important one because the majority of the SOD in vascular wall is attributed to SOD1.⁶ This is in line with the previous observation showing that superoxide anion levels are decreased in the aorta of SOD1 transgenic mice.^21,22 Most importantly, the stimulatory effect of EPO on expression of SOD1 has important therapeutic implications. In our previous studies we have shown that EPO increases expression of phosphorylated eNOS in wild-type mice.¹¹ Moreover, the local concentrations of NO in arterial wall are not only dependent on enzymatic activity of eNOS but are also determined by concentrations of superoxide anions.¹ Indeed, increased local production of superoxide anions appears to be an important component of the vessel response to injury. Consistent with this concept, previous studies have indicated that reactive oxygen species are important mediators of SMC proliferation and migration.²³ Likewise, superoxide production is increased not only as an early response of the vessel wall to injury, but also two weeks after injury.^13,24-26 Based on these observations, the current results suggest that the vascular protective effects of EPO in wild-type mice may be mediated at least in part via increased vascular wall SOD1 thereby protecting NO from inactivation by superoxide anions. The exact molecular mechanism responsible for activation of SOD1 by EPO remains to be determined.

To determine whether deletion of SOD1 gene may abolish vascular protective effects of EPO, vascular structure in injured carotid arteries of SOD1^-/- mice were studied. Quite unexpectedly, we found that under basal conditions, medial CSA of the carotid artery was significantly reduced in SOD1^-/- mice. This is at variance with the results reported by Baumbach and colleagues showing that vascular hypertrophy is present in cerebral arterioles of SOD1^-/- mice.²⁷ The cause of discrepancy is not unclear at the present time but could be related to the anatomical origin and functional differences between conduit arteries and cerebral arterioles. However, our findings also showed that vascular remodeling after wire-induced injury was less pronounced in SOD1^-/- mice as compared to wild-type mice. This result suggests that genetic inactivation of SOD1 may have previously unrecognized inhibitory effect on pathological remodeling after injury. Inactivation of SOD1 is known to increase intracellular concentrations of superoxide anions and peroxynitrite.⁶ In addition, we also showed that inactivation of SOD1 decreased release of H₂O₂ from the aorta. Such a decrease is relevant for interpretation of our findings, specifically because H₂O₂ plays an important role in the regulation of cell growth, proliferation, and development as well as in the progression of atherosclerosis.²⁸ Most importantly, H₂O₂ (but not the superoxide anion) enhances VSMC proliferation and growth.^29-31 Therefore, the reduced arterial wall thickness in SOD1^-/- mice could be explained by the absence of superoxide anion dismutation thereby significantly reducing vascular concentration of H₂O₂.³² In addition, we observed an increased protein expression of catalase in arterial wall of SOD1^-/- mice. This observation is in agreement with our suggestion that local concentration H₂O₂ is low in arteries of SOD1^-/- mice. Consistent with this interpretation of our findings, a recent study by Zhang et al demonstrated that overexpression of catalase decreases the hypertrophic effect of angiotensin II-induced hypertension¹⁹ while overexpression of SOD1 was ineffective²¹ thereby reinforcing the important role of H₂O₂ in control of vascular wall thickness in-vivo.

Akt is one of the most important molecular targets activated by physiological concentrations of H₂O₂.^33,34 It is also an important mediator in cell growth and survival.³⁵ Interestingly, phosphorylation of Akt1 at Ser⁴⁷³ was reduced in the aorta of SOD1^-/- mice indicating that the observed reduction in medial thickness may be caused by the decreased activity of Akt1.³⁶ Moreover, prior studies have demonstrated that arterial injury causes proliferation of VSMC via phosphorylation of Akt at Ser⁴⁷³.^37-40 The importance of Akt signaling is further emphasized by the results of in-vivo studies demonstrating that transduction of injured carotid artery with dominant-negative Akt mutant or treatment of animals with phosphatidylinositol 3-kinase inhibitor wortmannin, result in reduced VSMC proliferation.^36,37,40 Furthermore, physiological concentrations of H₂O₂ cause phosphorylation of Akt.⁴¹ Thus, it appears that reduced phosphorylation of Akt1 in SOD1^-/- mice, is likely consequence of low intracellular concentration of H₂O₂.

We also observed an increase of 3-nitrotyrosine positive proteins in SOD1^-/- mice, indicating an increased formation of the potent oxidant, peroxynitrite, by interaction of superoxide anion with NO. An excessive formation of peroxynitrite represents an important mechanism contributing to cell death and dysfunction in cardiovascular disease.⁴² Several in-vitro and in-vivo studies reported inhibition of Akt activity and Akt phosphorylation by peroxynitrite through a mechanism involving nitration and inactivation of phosphatidylinositol 3-kinase.^43-45 These mechanisms may also help to explain reduction of arterial wall thickness in SOD1^-/- mice.

Increased superoxide anions production contributes to endothelial dysfunction and pathogenesis of hypertension.⁴⁶ However, arterial blood pressure was paradoxically reduced in SOD1^-/- mice, despite presence of increased superoxide anions concentrations. The exact mechanism of hypotension induced by genetic inactivation of SOD1 is unclear. However, several studies suggest that decreased concentration of H₂O₂ may reduce SBP. Indeed, vascular specific overexpression of catalase resulted in reduction of blood pressure in mice⁴⁷ indicating that increased elimination of H₂O₂ in vascular wall may have hypotensive effecs. Moreover, the plasma concentration of H₂O₂ is increased in patients with essential hypertension and is positively correlated with SBP.⁴⁸ There is also evidence that superoxide anions can chemically inactivate catecholamines, thus lowering SBP in-vivo.⁴⁹ Although the exact mechanism of hypotension induced by genetic inactivation of SOD1 remains to be determined, the results of the present study offer new insights into alterations of arterial wall architecture in SOD1^-/- mice. We speculate that elevated concentrations of superoxide anions associated with low concentration of H₂O₂ and subsequent impairment of Akt signaling, may help to explain the lower arterial blood pressure observed in SOD1^-/- mice. On the other hand, we cannot rule out the possibility that changes in vascular architecture in SOD1^-/- mice are secondary to reduction in blood pressure caused by a mechanism that remains to be determined.

Traditionally, elevated concentration of superoxide anion and subsequent chemical inactivation of nitric oxide is considered detrimental for the vascular function.¹ Quite surprisingly, cardiovascular phenotypic characteristics of SOD1-deficient mice suggest that loss of SOD1 (and subsequent increase in vascular concentration of superoxide anion) may result in paradoxical hypotension and reduced propensity towards medial thickening. Despite the fact that these findings introduced difficulties in interpretation of our findings, it is important to re-emphasize that treatment with EPO significantly reduced production of superoxide anion and medial thickening in wild type mice. In contrast, elevated production of superoxide anion and significant thickening of injured arterial wall were not affected in SOD1-deficient animals thereby supporting major hypothesis of the present study.

Perspective

A growing body of evidence indicates that EPO has tissue-protective properties that are critically dependent both on increased eNOS and Akt activity and increased bioavailability of NO.^50,9-12 The present study showed that in wild-type mice, treatment with EPO increased vascular SOD1 expression and effectively prevented vascular remodeling after carotid artery injury. In contrast, genetic inactivation of SOD1 abolished ability of EPO to reduce concentrations of superoxide anions thereby suggesting that EPO exerts antioxidant effect in blood vessel wall by regulating expression and activity of SOD1 protein.