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(Stroke. 2005;36:2731.)
© 2005 American Heart Association, Inc.

Original Contributions

Role of Endothelial NO Synthase Phosphorylation in Cerebrovascular Protective Effect of Recombinant Erythropoietin During Subarachnoid Hemorrhage– Induced Cerebral Vasospasm

Anantha Vijay R. Santhanam, PhD; Leslie A. Smith; Masahiko Akiyama, MD; A. Gabriela Rosales, MS; Kent R. Bailey, PhD Zvonimir S. Katusic, MD, PhD

From the Departments of Anesthesiology and Molecular Pharmacology and Experimental Therapeutics (A.V.R.S., L.A.S., M.A., Z.S.K.) and the Division of Biostatistics (A.G.R., K.R.B.), Mayo Clinic College of Medicine, Rochester, Minn.

Correspondence to Zvonimir S. Katusic, Department of Anesthesiology, Mayo Clinic College of Medicine, 200 First St SW, Rochester, MN 55905. E-mail katusic.zvonimir{at}mayo.edu

	Abstract

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Background and Purpose— In the present study, the effect of subarachnoid hemorrhage (SAH) on the phosphorylation of endothelial NO synthase (eNOS) and the ability of recombinant erythropoietin (Epo) to augment this vasodilator mechanism in the spastic arteries were studied.

Methods— Recombinant adenoviral vectors (10⁹ plaque-forming units per animal) encoding genes for human Epo (AdEpo), and ß-galactosidase were injected immediately after injection of autologous arterial blood into the cisterna magna (day 0) of rabbits. Cerebral angiography was performed on day 0 and day 2, and basilar arteries were harvested for Western blots, measurement of cGMP levels, and analysis of vasomotor functions.

Results— Injection of autologous arterial blood into cisterna magna resulted in significant vasospasm of the basilar arteries. Despite the narrowing of arterial diameter and reduced expression of eNOS, expressions of phosphorylated protein kinase B (Akt) and phosphorylated eNOS were significantly increased in spastic arteries. Gene transfer of AdEpo reversed the vasospasm. AdEpo-transduced basilar arteries demonstrated significant augmentation of the endothelium-dependent relaxations to acetylcholine, whereas the relaxations to an NO donor, 2-(N,N-diethylamino)diazenolate-2-oxide sodium salt, were not affected. Transduction with AdEpo further increased the expression of phosphorylated Akt and eNOS and elevated basal levels of cGMP in the spastic arteries.

Conclusions— Phosphorylation of eNOS appears to be an adaptive mechanism activated during development of vasospasm. The vascular protective effect of Epo against cerebral vasospasm induced by SAH may be mediated in part by phosphorylation of Akt/eNOS.

Key Words: basilar artery • gene therapy • nitric oxide

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Phosphorylation of endothelial NO synthase (eNOS) is a key mechanism responsible for eNOS enzymatic activity and subsequent increase in production of NO.^1–5 Under physiological conditions, shear stress imposed on endothelial cells by circulating blood is critical stimulus maintaining eNOS in phosphorylated state.⁶ Pathogenesis of cerebral vasospasm induced by subarachnoid hemorrhage (SAH) is not completely understood; however, existing evidence suggests that reduced production or loss of biological activity of NO contributes to chronic narrowing of cerebral arteries surrounded by autologous blood.^7–9 The effect of SAH on phosphorylation of eNOS in the cerebral arteries has not been studied.

Attempts to develop effective vasoprotective strategies against cerebral vasospasm and subsequent brain ischemia have not been successful.¹⁰ More recent studies suggest that hypoxia-inducible cytokine erythropoietin (Epo) may have important nonhematopoietic functions, including neuroprotection.^11,12 Indeed, a recent clinical trial suggests that treatment with Epo has beneficial effect in patients with stroke.¹³ In addition to the effect on neuronal tissue, studies on experimental models of SAH suggest that Epo can prevent vasospasm;¹⁴ however, the exact mechanism(s) of vasoprotective effect is unknown. Recognized ability of Epo to activate protein kinase B (Akt) and subsequent phosphorylation of eNOS in cultured endothelial cells suggests that increased formation of NO could be an important mechanism underlying the therapeutic effect of Epo.^15–17 To date, no in vitro or in vivo study on intact arteries has been performed to determine the effect of Epo on eNOS phosphorylation. Thus, the present study was designed to test the hypothesis that the vasoprotective effect of Epo is mediated in part by phosphorylation of eNOS.

	Materials and Methods

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Animals
Male New Zealand white rabbits (2 to 3 kg) were used for experiments. Rabbits were anesthetized with 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 Sleepaway (3 mL; 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, white blood cell count, arterial pH, PO_2, and PCO₂.

Experimental Model of SAH
Rabbits were anesthetized and a 25-gauge needle was aseptically inserted into the cisterna magna, and 300 µL of cerebrospinal fluid (CSF) was withdrawn and replaced with 1 mL/kg of autologous arterial blood. After 30 minutes in the head-down position, the animals were allowed to recover. Forty-eight hours after injection, the animals were euthanized and the basilar arteries were isolated.

Adenoviral Vectors and Gene Transfer
Replication-incompetent adenovirus encoding the human Epo (AdEpo) gene and Escherichia coli ß-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 or SAH. A total of 300 µL of CSF was aspirated and mixed with 50 µL vector (10⁹ plaque-forming units [pfu]) or vehicle and injected aseptically using a 25-gauge needle into the cisterna magna immediately after injection of autologous arterial blood as described previously.^18,19 The transduction titer of 10⁹ pfu/rabbit was chosen based on previous in vivo gene transfer studies.^19,20 After injection, animals were maintained in a head-down position for 30 minutes before transfer to postanesthesia recovery.

Cerebral Angiography
Basilar artery angiography was performed on day 0 (baseline; before first injection of blood) and day 2 as described previously.¹⁹ A transfemoral catheter was advanced to the proximal vertebral artery, after which iodine radiocontrast was injected into the posterior cerebral circulation. The basilar artery was imaged radiographically and its diameter measured as described previously.¹⁹

Measurement of Epo Levels
Epo levels in the CSF and plasma were measured by a 2-site chemiluminescence immunoassay using the Nichols erythropoietin immunoassay kit (Nichols Institute Diagnostics) at the Endocrinology Laboratories of the Mayo Clinic.

Western Blot
Soluble proteins were extracted by mincing and homogenizing tissues in lysis buffer containing 50 mmol/L NaCl, 50 mmol/L NaF, 50 mmol/L sodium pyrophosphate, 5 mmol/L EDTA, 5 mmol/L EGTA, 0.1 mmol/L Na₃VO₄, 1% Triton X-100, 10 mmol/L HEPES, pH 7.4, and protease inhibitors (1.04 mmol/L 4-(2-aminoethyl)benzenesulfonyl fluoride, 15 µmol/L pepstatin A, 14 µmol/L E-64, 40 µmol/L bestatin, 20 µmol/L leupeptin, and 0.8 µmol/L aprotinin). Briefly, 50 µg protein was 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 antibodies (1:500 dilution) against phosphorylated S1177–eNOS (BD Transduction), Akt, and phosphorylated Akt (S473; Cell Signaling), polyclonal antibodies to eNOS (BD Biosciences), and Epo (Santa Cruz Biotechnology). Bands were visualized by enhanced chemiluminescence (Amersham Pharmacia).

Measurement of cGMP
Rabbit basilar arteries were incubated in DMEM in a CO₂ incubator at 37°C for 30 minutes in 3-isobutyl-1-methylxanthine (10^–4 mol/L; Sigma) to inhibit the degradation of cyclic nucleotides by phosphodiesterases. Then the rings were removed from the medium and quickly frozen in liquid nitrogen. After homogenization, cGMP levels were measured by a cGMP radioimmunoassay kit (Amersham).²¹ Protein assay was conducted by DC Protein Assay kit (Bio-Rad).

Analysis of Vascular Reactivity
Ring segments (3 mm long) were connected to an isometric force-displacement transducer (Grass FT03; Grass Instrument Co.) and suspended in an organ chamber filled with 25 mL of Krebs solution (composition [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% CO₂.²² Isometric force was recorded continuously. The rings were gradually stretched and allowed to stabilized at a resting force of 500 mg for 45 minutes. Rabbit basilar arteries were contracted with median EC₅₀ of histamine (3x10^–7 to 1x10^–6 mol/L) before the cumulative addition of either acetylcholine (10^–9 mol/L to 10^–5 mol/L) or 2-(N,N-diethylamino)diazenolate-2-oxide sodium salt (DEA-NONOate; 10^–9 mol/L to 10^–5 mol/L) to obtain the relaxation responses.

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±SEM for n (n=No. of rabbits in each group) animals used in each experimental group. Relaxations are expressed as percentage of maximal relaxations induced by 3x10^–4 mol/L papaverine. cGMP values were analyzed by unpaired Student t test. Densitometric value comparisons across different groups were assessed by 1-way ANOVA followed by pairwise comparisons. Differences among relaxation values across concentration-response curves were analyzed by 2-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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Biological Parameters
In vivo gene delivery did not affect the counts of red blood cells, white blood cells, hemoglobin, or hematocrit (Table). During angiography, the pH, PO₂, and PCO₂ of the arterial blood gases were monitored and were not different after SAH or after adenoviral gene delivery (data not shown). The CSF and plasma levels of Epo were significantly increased in the AdEpo-transduced group after SAH compared with either control or AdLacZ-transduced spastic rabbits (Table). 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. This observation could be attributable to the nonspecific binding of the human antibodies to proteins present in the rabbit CSF.

View this table: [in this window] [in a new window]   Hematological Parameters and Epo Levels

Gene Transfer of Epo to Basilar Arteries
Subsequent to gene transfer of AdEpo, protein expression of Epo was detected by Western blot only in AdEpo-transduced spastic basilar arteries (Figure 1).

View larger version (22K): [in this window] [in a new window]   Figure 1. Representative Western blot and densitometric analysis demonstrating Epo expression in spastic basilar arteries subsequent to intracisternal AdEpo or AdLacZ gene transfer. A total of 50 µg of protein obtained from a single basilar artery was loaded in each lane. Data are presented as means±SEM (n=3 rabbits in each group).

Effect of Epo on Vasospasm After SAH
Rabbits subjected to SAH demonstrated significant vasospasm 2 days after injection of autologous arterial blood, with 52% reduction (1.09±0.11 mm in control versus 0.48±0.03 mm in SAH; P<0.01; n=11) in the mean diameter of the basilar arteries exposed to SAH (Figure 2A).^14,19,23 Rabbits transduced with AdEpo showed a significant reversal of vasospasm, whereas transduction with AdLacZ did not affect vasospasm subsequent to SAH (Figure 2A and 2B).

View larger version (68K): [in this window] [in a new window]   Figure 2. A, Representative angiograms of the basilar arteries of control rabbits or rabbits subjected to SAH alone or SAH followed by intracisternal injection with AdLacZ or AdEpo. B, Bar diagram representing the average diameter of the basilar arteries from control rabbits or rabbits subjected to SAH or SAH followed by intracisternal injection with AdLacZ or AdEpo. *Differences in the average diameter of the basilar artery among control and SAH or SAH+AdLacZ are significant (P<0.01; n=6 to 11, respectively). #Differences in the average diameter of the basilar artery among SAH and SAH+AdEpo group is statistically significant (P<0.05; n=4 to 6).

Effect of Epo on Vasomotor Functions of SAH Rabbit Basilar Arteries
As reported previously by Kim et al (2003),²³ endothelium-dependent relaxations to acetylcholine in rabbit basilar artery were not affected by SAH. However, relaxations to acetylcholine were significantly augmented in basilar arteries transduced with AdEpo compared with AdLacZ-transduced spastic basilar arteries (Figure 3A). Endothelium-independent relaxations to NO donor DEA-NONOate (10^–9 mol/L to 10^–5 mol/L) were not different among control, AdLacZ- or AdEpo-transduced spastic basilar arteries (Figure 3B). Also, gene transfer of AdEpo significantly elevated the levels of cGMP in SAH rabbits compared with SAH rabbits transduced with AdLacZ (Figure 4).

View larger version (19K): [in this window] [in a new window]   Figure 3. A, Endothelium-dependent relaxations to acetylcholine in basilar arteries of control rabbits and rabbits subjected to SAH followed by gene transfer with AdLacZ or AdEpo. Relaxations were obtained during contractions induced by histamine (3x10–7 mol/L to 1x10–6 mol/L). Data are expressed as percentage of maximal relaxation induced by 3x10–4 mol/L papaverine; 100%=1.34±0.19 g, 1.10±0.07 g, 0.96±0.10 g in control, SAH+AdLacZ and SAH+AdEpo arteries, respectively (n=5). *Significantly different in SAH+AdEpo compared with SAH+AdLacZ (P<0.05). B, Endothelium-independent relaxations to DEA-NONOate in basilar arteries of control rabbits and rabbits subjected to SAH followed by gene transfer with AdLacZ or AdEpo. Relaxations were obtained during contractions induced by histamine (3x10–7 mol/L to 1x10–6 mol/L). Data are expressed as percentage of maximal relaxation induced by 3x10–4 mol/L papaverine; 100%=1.19±0.11 g, 1.08±0.06 g, 1.04±0.10 g in control, SAH+AdLacZ, and SAH+AdEpo arteries, respectively (n=5).

View larger version (12K): [in this window] [in a new window]   Figure 4. Bar diagram representing the levels of basal cGMP among basilar arteries of control or rabbits subjected to SAH or SAH rabbits transduced with AdLacZ or AdEpo. *Differences in the levels of cGMP among control or SAH and SAH+AdEpo are statistically significant (P<0.05; n=5).

Mechanisms of Vascular Protection by Epo
The expression of eNOS was significantly attenuated in arteries subjected to SAH (Figure 5A). However, the expression of phosphorylated form of eNOS was significantly upregulated in rabbits subjected to SAH. Gene transfer mediated by AdEpo further potentiated this increase in the expression of phosphorylated eNOS (Figure 5B). The expression of protein kinase B (Akt), a major activator of eNOS enzymatic activity, was not significantly affected by SAH or gene delivery (Figure 5C). However, SAH induced increase of phosphorylated Akt expression. This effect was further increased in the AdEpo-transduced basilar arteries (Figure 5D).

View larger version (26K): [in this window] [in a new window]   Figure 5. A, Representative Western blot and densitometric analysis of the protein expression of eNOS among control, rabbits subjected to SAH alone, or SAH rabbits transduced with AdLacZ or AdEpo. *Expression of eNOS is significantly lower in SAH alone or SAH+AdLacZ or SAH+AdEpo–basilar arteries compared with control (P<0.05; n=4). B, Representative Western blot and densitometric analysis of the protein expression of phosphorylated eNOS among control, rabbits subjected to SAH alone, or SAH rabbits transduced with AdLacZ or AdEpo. *Expression of phosphorylated eNOS is significantly higher in SAH alone or SAH+AdLacZ or SAH+AdEpo–basilar arteries compared with control (P<0.05; n=4 to 6). #Expression of phosphorylated eNOS is further potentiated (P<0.05; n=4 to 6) in SAH+AdEpo–basilar arteries compared with spastic controls. C, Representative Western blot and densitometric analysis of the protein expression of Akt among control, rabbits subjected to SAH alone, or SAH rabbits transduced with AdLacZ or AdEpo. D, Representative Western blot and densitometric analysis of the protein expression of phosphorylated Akt among control, rabbits subjected to SAH alone, or SAH rabbits transduced with AdLacZ or AdEpo. *Expression of phosphorylated Akt is significantly higher in SAH alone or SAH+AdLacZ or SAH+AdEpo–basilar arteries compared with control (P<0.05; n=4 to 6). #Expression of phosphorylated Akt is further potentiated (P<0.05; n=4 to 6) in SAH+AdEpo–basilar arteries compared with spastic controls.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
This is the first in vivo study to provide insight into the molecular mechanisms underlying protective vascular effect of recombinant Epo in cerebral circulation. We report several novel findings. First, expression of eNOS protein is reduced, whereas levels of phosphorylated eNOS protein and phosphorylated protein kinase B (Akt) are increased in spastic arteries. Second, phosphorylation of eNOS and protein kinase B is robustly amplified in spastic cerebral arteries treated with recombinant Epo. Third, perivascular gene delivery of recombinant Epo prevents vasospasm induced by SAH, suggesting that phosphorylation of Akt and eNOS are indeed important molecular mechanisms responsible for the beneficial effect of Epo.

In vivo reduction in arterial diameter increases blood flow velocity²⁴ and shear stress,²⁵ a known activator of the phosphoinositide-3-kinase/protein kinase B (Akt) and subsequent phosphorylation of eNOS.^1–6 Findings of the present study are the first to suggest that phosphorylation of eNOS could be an important adaptive mechanism designed to preserve NO biosynthesis in early stages of vasospasm characterized by the loss of eNOS protein. We also provide evidence that phosphorylation of Akt is the most likely upstream event responsible for phosphorylation of eNOS. These observations are important because they indicate that eNOS phosphorylation could be exploited as molecular target for therapeutic intervention. To test this concept, we analyzed the effect of recombinant Epo on cerebral vasospasm. Epo was chosen because previous studies on cultured endothelial cells demonstrated that it can stimulate phosphorylation of Akt and eNOS.²⁶ In addition, Epo has demonstrated neuroprotective effect in experimental models of stroke as well as in patients with acute stroke.^{12–15,27–30}

Adenovirus-mediated gene transfer was used to deliver recombinant Epo into cisterna magna. Several previous studies from our group demonstrated that this approach results in efficient gene delivery into the large cerebral arteries.^19–22 Indeed, successful gene transfer of Epo was confirmed by the increased levels of Epo in CSF and by the presence of recombinant Epo in the AdEpo-transduced basilar arteries. In addition, we detected high circulating levels of Epo after intracisternal AdEpo delivery. The crossing of recombinant Epo from CSF into circulating blood was the most likely reason for elevation of Epo in circulating blood. Alternatively, intracisternal delivery of AdEpo could result in expression, production, and release of Epo from a peripheral tissue inadvertently transduced by adenovirus. The results of the present study cannot rule out this possibility. Thus, observed changes in cerebral arteries of rabbits transduced by AdEpo are a result of exposure to high intraluminal and perivascular concentration of recombinant Epo. However, it is important to note that the circulating concentration of Epo in rabbits transduced with AdEpo was comparable to the concentration detected in patients treated with intravenous injections of Epo during stroke,¹⁴ suggesting that in the present study, circulating levels of Epo were within the clinically reported therapeutic range.¹⁴

Several lines of evidence support our conclusion that beneficial effect of recombinant Epo resulting in prevention of vasospasm is mediated by increased production of endothelial NO. Enhanced endothelium-dependent relaxations were observed in AdEpo-transduced spastic basilar arteries. Endothelium-dependent relaxations of rabbit basilar arteries to acetylcholine are mediated primarily by increased production and release of NO and were significantly augmented by AdEpo gene delivery. In contrast, relaxations to endothelium-independent vasodilator NONOate were not affected by AdEpo gene delivery, suggesting that the effect of Epo was selective for endothelial cells. Consistent with enhancement of endothelium-dependent relaxations, AdEpo-transduced arteries had significantly increased levels of cGMP, the second messenger for NO. Although we did not directly measure production of NO in basilar artery, elevation of cGMP in arterial wall is certainly consistent with high local concentration of NO. Most notably, expression of phosphorylated eNOS protein was increased after treatment with recombinant Epo, reinforcing our conclusion regarding the importance of eNOS phosphorylation in prevention of vasospasm induced by SAH. However, we cannot rule out that other mechanisms, including intracellular localization of eNOS as well as local concentrations of eNOS substrate, L-arginine, and eNOS cofactor tetrahydrobiopterin, may contribute to the beneficial effect of Epo.

Two days after SAH, cerebral vasospasm in rabbits correspond to the early mildest form of vasospasm in humans.³¹ Furthermore, unlike in chronic severe vasospasm in humans, vasospasm in rabbits is caused mainly by contraction of smooth muscle cells that can be reversed by vasodilators including papaverine.^32,33 However, despite these limitations, rabbit model can provide important information concerning early adaptive (or maladaptive) changes in cerebral arterial wall exposed to autologous blood. The results of the present study demonstrate that phosphorylation of eNOS is an important early event in pathogenesis of vasospasm. Whether compounds capable of enhancing this response to SAH may have therapeutic value in treatment of chronic severe vasospasm remains to be determined. Epo is certainly an attractive therapeutic molecule because of its protective effect on vascular and neuronal function.^{12,13,15,28–31}

Results of the present study demonstrate vasoprotective effect of Epo in prevention and treatment of cerebral vasospasm. Gene transfer of Epo subsequent to SAH increased the phosphorylation of Akt and eNOS, thereby increasing NO production in the basilar arteries. We speculate that in addition to its previously reported protective effects on the neurons, Epo enhances the vasoprotective mechanisms by upregulating the expression and function of eNOS-derived NO.

	Acknowledgments

 
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, and the Mayo Foundation.

Received June 1, 2005; revision received September 6, 2005; accepted September 23, 2005.

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