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Am J Physiol Heart Circ Physiol 291: H781-H786, 2006. First published March 24, 2006; doi:10.1152/ajpheart.00045.2006
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In vivo stimulatory effect of erythropoietin on endothelial nitric oxide synthase in cerebral arteries

Anantha Vijay R. Santhanam,1,2 Leslie A. Smith,1,2 Karl A. Nath,3 and Zvonimir S. Katusic1,2

1Departments of Anesthesiology, and 2Molecular Pharmacology and Experimental Therapeutics, and 3Division of Nephrology and Hypertension, Mayo Clinic College of Medicine, Rochester, Minnesota

Submitted 9 January 2006 ; accepted in final form 4 March 2006


   ABSTRACT
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 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
The discovery of tissue protective effects of erythropoietin has stimulated significant interest in erythropoietin (Epo) as a novel therapeutic approach to vascular protection. The present study was designed to determine the cerebral vascular effects of recombinant Epo in vivo. Recombinant adenoviral vectors (109 plaque-forming units/animal) encoding genes for human erythropoietin (AdEpo) and beta-galactosidase (AdLacZ) were injected into the cisterna magna of rabbits. After 48 h, basilar arteries were harvested for analysis of vasomotor function, Western blotting, and measurement of cGMP levels. Gene transfer of AdEpo increased the expressions of recombinant Epo and its receptor in the basilar arteries. Arteries exposed to recombinant Epo demonstrated attenuation of contractile responses to histamine (10–9 to 10–5 mol/l) (P < 0.05, n = 5). Endothelium-dependent relaxations to acetylcholine (10–9 to 10–5 mol/l) were significantly augmented (P < 0.05, n = 5), whereas endothelium-independent relaxations to a nitric oxide (NO) donor 2-(N,N-diethylamino)diazenolate-2-oxide sodium salt remained unchanged in AdEpo-transduced basilar arteries. Transduction with AdEpo increased the protein expression of endothelial NO synthase (eNOS) and phosphorylated the S1177 form of the enzyme. Basal levels of cGMP were significantly elevated in arteries transduced with AdEpo consistent with increased NO production. Our studies suggest that in cerebral circulation, Epo enhances endothelium-dependent vasodilatation mediated by NO. This effect could play an important role in the vascular protective effect of Epo.

recombinant adenoviral vectors; vasodilatation; gene transfer; rabbit

ERYTHROPOIETIN (Epo) was originally described as a regulator of erythropoiesis (26). However, the biological effects of Epo are not restricted to erythroid precursor cells. Epo can also affect the functions of endothelial, vascular smooth muscle, myocardial, mesangial, and neuronal cells (4–6, 19, 32, 34, 45). Epo modulates a broad array of cellular processes that include progenitor stem cell development, cellular integrity, and angiogenesis (31).

Recently, several studies have demonstrated a significant neuroprotective effect of Epo and its nonhematopoietic derivatives (9, 17, 20, 27), and a proof of concept clinical trial demonstrated beneficial effects of Epo in patients with an acute stroke (16). The concentration of Epo used to demonstrate therapeutic efficacy of Epo in experimental models of neurological disorders as well as in patients with acute stroke is about 500- to 1,000-fold higher than the circulating levels of Epo (16, 28). However, the effect of such high circulating, but therapeutically effective, levels of Epo on normal cerebral vasculature has not been studied.

Epo is reported to increase endothelial nitric oxide (NO) synthase (eNOS) expression and NO production in cultured endothelial cells (7). Administration of Epo has also been reported to increase eNOS expression in vivo in rats (23). However, long-term administration of Epo in patients is associated with hypertension (18, 24, 37), in part, mediated by either increased intracellular calcium in smooth muscle cells (43) or increased endothelin production (10, 11). In addition, Epo can impair endothelium-dependent vasodilatation in humans, probably through a cyclooxygenase-dependent mechanism (44). Hence, divergent effects of Epo on the vasculature have been reported to date. In a recent study, we observed that Epo increased the phosphorylation of eNOS and reversed cerebral vasospasm rabbits subjected to subarachnoid hemorrhage (39). From these the findings, we hypothesized that in the cerebral vasculature, Epo increases the expression and activation of eNOS and augments vasodilatation.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. Male New Zealand White rabbits (2–3 kg) were used for experiments. Rabbits were anesthetized with an intramuscular injection composed of ketamine (35 mg/kg), xylazine (5 mg/kg), and acepromazine (2.3 mg/kg). Animals were anesthetized and euthanized on day 2 with intravenous pentobarbital sodium (Sleepaway, 260 mg/kg, Fort Dodge Animal Health). All procedures were approved by the Institutional Animal Care and Use Committee. Biological parameters measured in animals included heart rate, hematocrit, hemoglobin, erythrocyte count, and white blood cell count.

Adenoviral vectors and gene transfer. Replication-incompetent adenovirus encoding the human erythropoietin (AdEpo) gene and Escherichia coli beta-galactosidase (AdLacZ; vector-control in this study) were obtained from the Vector Core of the University of Pittsburgh. Control rabbits or arteries refer to those not exposed to vector. Cerebrospinal fluid (CSF, 300 µl) was aspirated and mixed with 50 µl vector [109 plaque-forming units (pfu)] or vehicle and injected aseptically by using a 25-gauge needle into the cisterna magna (25, 36). The transduction titer of 109 pfu/rabbit was chosen based on previous in vivo gene transfer studies (12, 25). After injection, animals were maintained in a head-down position for 30 min before transfer to postanesthesia recovery.

Measurement of EPO levels. Epo levels in the CSF and plasma were measured by a two-site chemiluminescence immunoassay using the Nichols Erythropoietin Immunoassay kit (Nichols Institute Diagnostics, San Clemente, CA) as described elsewhere (39).

Western blot analysis. Soluble proteins were extracted by mincing and homogenizing tissues in lysis buffer as described earlier (39). Briefly, 50 µg protein were separated by electrophoresis and transferred onto nitrocellulose membrane. Ponceau S staining of the membrane was performed to confirm equal loading subsequent to transfer. Blots were incubated with monoclonal antibody (1:500 dilution) against inducible NOS, phosphorylated S1177-eNOS (BD Transduction), actin (Santa Cruz), Akt and phosphorylated S473-Akt (Cell Signaling), heme oxygenase-1 (HO-1, Stressgen), and polyclonal antibodies to eNOS (BD Biosciences), Epo, and EpoR (Santa Cruz). Bands were visualized by enhanced chemiluminescence (Amersham Pharmacia).

Analysis of vascular reactivity. Ring segments (~3 mm long) were connected to an isometric force-displacement transducer (Grass FT03; Grass Instrument) and suspended in an organ chamber filled with 25 ml of Krebs solution (in mmol/l: 118.3 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 25 NaHCO3, 0.026 calcium-ethylenediaminetetracetic acid, and 11.1 glucose, 37°C, pH 7.4) aerated with 94% O2-6% CO2 (13). Isometric force was recorded continuously. The rings were gradually stretched and allowed to stabilize at a resting force of 500 mg for 45 min. Rabbit basilar arteries were contracted with histamine (3 x 10–7 to 1 x 10–6 mol/l) (21, 34, 38) before the cumulative addition of either acetylcholine (10–9 to 10–5 mol/l) or 2-(N,N-diethylamino)diazenolate-2-oxide sodium salt (DEA-NONOate; 10–9 to 10–5 mol/l) to obtain the relaxation responses.

Measurement of cGMP and cAMP. Rabbit basilar arteries were incubated in minimum essential medium in a CO2 incubator at 37°C for 30 min in 3-isobutyl-1-methylxanthine (IBMX, 10–3 mol/l; Sigma) to inhibit the degradation of cyclic nucleotides by phosphodiesterases. The rings were then removed from the medium and quickly frozen in liquid nitrogen. After homogenization, cGMP and cAMP levels were measured by cGMP and cAMP radioimmunoassay kits, respectively (Amersham) (3). Protein assay was conducted by DC Protein Assay kit (Bio-Rad).

Drugs. DEA-NONOate was obtained from Cayman Chemical. All other drugs used in the study were obtained from Sigma. Concentrations of all drugs are expressed as the final moles per liter in the organ chambers.

Statistical analysis. Results of the study are expressed as means ± SE of the number of rabbits in each group (n) used in each experimental group. Relaxations are expressed as percentage of maximal relaxations induced by 3 x 10–4 mol/l papaverine. cGMP values were analyzed by unpaired Student's t-test. Densitometric value comparisons across different groups were assessed by one-way ANOVA followed by pairwise comparisons. Differences among relaxation values across concentration-response curves were analyzed by two-way ANOVA followed by pairwise comparisons. Multiple comparisons adjustment was performed by Bonferroni method. A P value <0.05 was considered statistically significant.


   RESULTS
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DISCUSSION
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Physiological parameters. In vivo gene delivery did not affect the counts of red blood cells (RBCs), white blood cells (WBCs), hemoglobin, or hematocrit after 2 days (Table 1). TheCSF and plasma levels of Epo were significantly increased in the AdEpo-transduced group compared with either control or AdLacZ-transduced rabbits (Table 1). We do not have an explanation for the 10-fold higher levels of Epo detected in the CSF compared with the levels in plasma under basal conditions. We speculate that this observation could be attributable to the nonspecific binding of the human antibodies to proteins present in the rabbit CSF.


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  		Table 1. Physiological parameters in rabbits 2 days after gene delivery

 
Gene transfer of erythropoietin to basilar arteries. Subsequent to gene transfer, as shown by Western blot analysis, protein expression of Epo and EpoR were detected or increased respectively only in AdEpo-transduced basilar arteries (Fig. 1).


Figure 1
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  		Fig. 1. Representative Western blot analysis of three experiments demonstrating protein expression of erythropoietin (Epo) and its receptor in basilar arteries subsequent to intracisternal human Epo (AdEpo) or beta-galactosidase (AdLacZ) gene transfer. Fifty micrograms of protein obtained from a single basilar artery were loaded in each lane.

 
Effect of Epo on vasomotor function of rabbit basilar arteries. Concentration-dependent contractions to histamine (10–9 to 10–5 mol/l) were significantly reduced in AdEpo-transduced basilar arteries (Fig. 2). Endothelium-dependent relaxations to acetylcholine were significantly augmented in basilar arteries transduced with AdEpo compared with AdLacZ-transduced basilar arteries (Fig. 3A). Endothelium-independent relaxations to a NO donor DEA-NONOate (10–9 to 10–5 mol/l) were not different among control, AdLacZ-, or AdEpo-transduced basilar arteries (Fig. 3B).


Figure 2
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  		Fig. 2. Concentration-dependent contractions to histamine in basilar arteries of rabbits subjected to AdLacZ or AdEpo gene transfer. *Differences in contractions among AdLacZ and AdEpo are statistically significant (P < 0.05, n = 5 rabbits).

 

Figure 3
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  		Fig. 3. A: endothelium-dependent relaxations to acetylcholine in basilar arteries of rabbits subjected to AdLacZ or AdEpo gene transfer. Relaxations were obtained during contractions induced by histamine (3 x 10–7 to 1 x 10–6 mol/l). Data are expressed as percentage of maximal relaxation induced by 3 x 10–4 mol/l papaverine; 100% = 1.28 ± 0.17 g, 1.02 ± 0.11 g in AdLacZ and AdEpo arteries, respectively (n = 5 rabbits). *Significantly different in AdEpo compared with AdLacZ (P < 0.05). B: endothelium-independent relaxations to 2-(N,N-diethylamino)diazenolate-2-oxide sodium salt (DEA-NONOate) in basilar arteries of rabbits subjected to AdLacZ or AdEpo gene transfer. Relaxations were obtained during contractions induced by histamine (3 x 10–7 to 1 x 10–6 mol/l). Data are expressed as percentage of maximal relaxation induced by 3 x 10–4 mol/l papaverine; 100% = 1.33 ± 0.22 g, 1.06 ± 0.13 g in AdLacZ and AdEpo arteries, respectively (n = 5 rabbits).

 
In AdEpo-transduced arteries, the expressions of eNOS (Fig. 4A) and phosphorylated S1177-eNOS (Fig. 4B) were significantly elevated compared with AdLacZ-transduced arteries, whereas the expressions of iNOS (Fig. 4C) and HO-1 (Fig. 4D) remained unchanged. Furthermore, the expression of phosphorylated S473-Akt was significantly increased in AdEpo-transduced arteries, whereas the expression of Akt remained unchanged (Fig. 5).


Figure 4
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  		Fig. 4. A: representative Western blot analysis and densitometric analysis of protein expression of endothelial nitric oxide synthase (eNOS) in AdLacZ- and AdEpo-transduced basilar arteries. **Expression of eNOS is significantly increased in AdEpo-basilar arteries compared with AdLacZ-transduced arteries (P < 0.05; n = 5 rabbits). B: representative Western blot analysis and densitometric analysis of the protein expression of phosphorylated (S1177) eNOS in AdLacZ- and AdEpo-transduced basilar arteries. *Expression of phosphorylated eNOS is significantly higher in AdEpo-transduced basilar arteries compared with AdLacZ-transduced arteries (P < 0.05; n = 4–6 rabbits). C: representative Western blot analysis and densitometric analysis of the protein expression of inducible nitric oxide synthase (iNOS) in AdLacZ- or AdEpo-transduced basilar arteries (n = 4 rabbits). D: representative Western blot analysis and densitometric analysis of the protein expression of heme oxygenase-1 (HO-1) in AdLacZ- or AdEpo-transduced basilar arteries (n = 3 rabbits).

 

Figure 5
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  		Fig. 5. Representative Western blots analysis of Akt and phosphorylated S473-Akt and densitometric analysis of phosphorylated S473-Akt expression in AdLacZ- and AdEpo-transduced basilar arteries. *Expression of phosphorylated S473-Akt is significantly higher in AdEpo-transduced basilar arteries compared with AdLacZ-transduced arteries (P < 0.05; n = 3–4 rabbits).

 
Gene transfer of AdEpo significantly elevated the basal levels of cGMP compared with rabbits transduced with AdLacZ (Fig. 6A). This effect was selective for cGMP because levels of cAMP were not affected by Epo (Fig. 6B).


Figure 6
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  		Fig. 6. A: levels of basal cGMP among basilar arteries of rabbits transduced with AdLacZ or AdEpo. *Differences in the levels of cGMP among AdLacZ and AdEpo are statistically significant (P < 0.05, n = 5–8 rabbits). B: levels of basal cAMP among basilar arteries of rabbits transduced with AdLacZ or AdEpo.

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
This is the first in vivo study to examine the effects of recombinant Epo on normal cerebral arteries. Our study reports several novel findings. In cerebral arteries treated with recombinant Epo, contractile responses to histamine were attenuated, whereas endothelium-dependent relaxations to acetylcholine were significantly augmented. Furthermore, perivascular gene delivery of recombinant Epo increases the expression of eNOS, phosphorylated S1177-eNOS, and phosphorylated S473-Akt and elevates cGMP in the vasculature, thereby suggesting that exposure of cerebral arteries to recombinant Epo augments endothelial production of NO.

In the present study, experiments were performed on the basilar artery primarily because of its proximity to the site of gene delivery, i.e., cisterna magna. In a series of previous studies, we demonstrated that intracisternal gene delivery resulted in expression of recombinant protein in adventitial fibroblasts of cerebral arteries (3, 12, 13, 25, 36, 42). In the present study, successful gene transfer was confirmed by the increased levels of Epo in CSF and by the detection of Epo in the AdEpo-transduced basilar arteries. We also detected an increase in circulating levels of Epo. This observation could be explained by the crossing of Epo from CSF into the systemic circulation. Less likely, but another possible explanation involves transduction of peripheral tissues by recombinant AdEpo translocated from the CSF to systemic circulation. We want to point that the circulating levels of Epo were comparable to the concentration detected in patients treated with intravenous injections of Epo during an acute stroke (16). To the best of our knowledge, this is the first report to demonstrate the vascular effect of Epo at concentrations, which in clinical trials, exert beneficial effects in patients with an acute stroke. The increased expression of EpoR in Epo-transduced blood vessels suggests that in cerebral arteries, EpoR expression is regulated by Epo, in part, by reduced EpoR degradation as proposed by Martinez-Estrada and colleagues (33). Our results are also in agreement with previous reports on EpoR regulation in cultured endothelial cells and neurons (7, 14) and provide an additional insight into the vascular protective and neuroprotective mechanism(s) of recombinant Epo.

In an earlier study, we reported that the cerebral vascular protective effect of recombinant Epo during subarachnoid hemorrhage-induced cerebral vasospasm is mediated, in part, by augmentation of endothelium-dependent relaxations, increased phosphorylation of eNOS, and elevation of cGMP levels in the cerebral arteries (39). On the basis of these observations, we first examined the effect of Epo on contractions and relaxations in AdEpo-transduced arteries. In the present study, we expanded our previous findings and demonstrated that in normal cerebral arteries, expression of recombinant Epo attenuates contractile responses to histamine. The exact mechanism responsible for reduced contractions to histamine is unknown but may include increased basal production of NO or activation of eNOS by histamine (29, 41). Furthermore, we observed augmentation of endothelium-dependent relaxations to acetylcholine, whereas the smooth muscle reactivity to NO remained unaffected, suggesting that the endothelium is major target for cerebral vascular effect of Epo.

Recombinant human Epo is known to exert vasoconstrictor effects on isolated renal and mesenteric resistance vessels (22), and high concentrations of Epo have been shown to induce contractions of rat mesangial and aortic smooth muscle cells (34). In cultured endothelial cells, recombinant Epo may reduce expression of eNOS, whereas in vascular smooth muscle cells Epo raises the cytosolic calcium content of vascular smooth muscle cells by stimulation of protein kinase C and phospholipase C{gamma}1 (2, 43, 46). However, in these studies, the concentrations of Epo used (20–250 U/ml) greatly exceeded concentrations observed in plasma after adenoviral-mediated gene transfer of recombinant Epo in our study (~5 U/ml; Table 1). Our findings demonstrate that in cerebral arteries, recombinant Epo increased the phosphorylation of Akt and eNOS, resulting in augmentation of endothelium-dependent relaxation to acetylcholine. Indeed, endothelium-dependent relaxations to acetylcholine in rabbit basilar arteries are predominantly mediated by NO (30, 40). The lack of potentiation of relaxations by an endothelium-independent vasodilator DEA-NONOate suggests that the effect of Epo is selective for endothelial cells.

High basal levels of cGMP detected in Epo-transduced arteries most likely reflect high enzymatic activity of eNOS and increased NO production, consistent with Epo-induced increase in expression of eNOS. The protein expressions of iNOS and HO-1 remained unchanged in Epo-transduced arteries, reinforcing our conclusion that the effect of Epo is mediated by enhanced endothelial NO production in cerebral arteries. This is in line with the results obtained on transgenic mice overexpressing human Epo (38), wherein increased eNOS synthase expression, enhanced NO-mediated endothelium-dependent relaxation, and elevated circulating and vascular tissue NO were observed. In contrast to Epo-transgenic mice, the contribution of shear stress to stimulation of eNOS activity (8, 15) in cerebral arteries after exposure to Epo is unlikely, because we did not observe an increase in RBC number 2 days after adenoviral gene transfer.

As the clinical investigation into the application of recombinant Epo in stroke is gaining impetus, the multiple paracrine/autocrine functions of this hematopoietic hormone are emerging. In addition to neuroprotection, Epo exerts protection against ischemic injury in the heart and induces angiogenesis in vivo. Our results provide the first in vivo evidence that recombinant Epo has a direct action on the endothelium and increases NO bioavailability by upregulation of eNOS in cerebral circulation. The findings of the present study offer an insight into the potential mechanism(s) underlying the vascular protection by Epo.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This work was supported in part by National Heart, Lung, and Blood Institute Grants HL-53524, HL-58080, and HL-66958, the American Heart Association Bugher Award for Investigation of Stroke, American Heart Association Postdoctoral Fellowship (AVRS), and the Mayo Foundation.


   ACKNOWLEDGMENTS
 
The authors thank Janet Beckman for assistance with preparation of this manuscript.

Part of this work has been presented as a poster at Experimental Biology 2005 in San Diego, CA.


   FOOTNOTES
 

Address for reprint requests and other correspondence: Z. S. Katusic, Dept. of Anesthesiology, Mayo Clinic College of Medicine, 200 First St. SW, Rochester, MN 55905 (e-mail: katusic.zvonimir{at}mayo.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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