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1 Digestive Research Laboratory, Fundación Jiménez Díaz2 Cardiovascular Research Unit3 Nephrology Department, Hospital Clínico San Carlos, Madrid 28040, Spain
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Abstract |
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(Received 28 January 2005;
accepted after revision 4 March 2005; first published online 15 March 2005)
Corresponding author A. J. López-Farré: Cardiovascular Research Unit, Hospital Clínico San Carlos, C/Profesor Martín Lagos s/n, Madrid 28040, Spain. Email: ajlopez.hcsc{at}salud.madrid.org
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Introduction |
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Nitric oxide (NO) is generated by the endothelium through the activity of the endothelial nitric oxide synthase (eNOS) (Moncada et al. 1991). In this regard, the lack of the NO-dependent relaxation may be caused by a host of factors which induce endothelial dysfunction in humans and experimental animal models. Indeed, loss of the ability of the endothelium to release NO, which has been termed endothelial dysfunction, results in vascular abnormalities including vasoconstriction, platelet activation and adhesion of blood elements to the endothelial surface (Moncada & Higgs, 1993; López-Farré et al. 1995), effects that are also produced during hypoxic–reperfusion (HR) injury (Moncada, 1993). Endothelial dysfunction is a ubiquitous finding after reduction of oxygen flow to various tissues including the kidney. Therefore, endothelial hypoxic preconditioning may be an attractive goal to prevent vascular injury produced by HR. In this regard, although the mechanisms implicated in the induction of endothelial dysfunction during HR are not yet delineated, the reduction of the early endothelial dysfunction associated with HR through the maintenance of an adequate level of eNOS activity could be important.
eNOS activity is highly regulated and recent findings have demonstrated that serine1177 phosphorylation of eNOS protein leads to eNOS activation adding more complexity to the post-translational regulation of eNOS (Dimmeler et al. 1999). It is also now recognized that protein–protein interactions of eNOS protein with chaperone heat shock protein 90 (hsp90) facilitates phosphorylation of serine1177 (Garcia-Cardena et al. 1998). In this sense, an increased amount of hsp90 coprecipitating with eNOS protein has been associated with increased NO production by the endothelium (Garcia-Cardena et al. 1998). Indeed, Fontana et al. (2002) have recently demonstrated the importance of hsp90–eNOS interactions in the induction of eNOS phosphorylation and eNOS activation. However, to our knowledge, the possibility that hypoxic preconditioning may affect hsp90-dependent regulation of eNOS activity and thus protect endothelial functionality has not yet been explored. Therefore, the aim of the present study was to analyse the level of both serine1177 phosphorylation of eNOS and the interaction between hsp90 and eNOS protein in the vascular wall of an in vitro model of early hypoxic preconditioning developed in isolated rat aortic segments.
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Methods |
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The study was performed in male Wistar rats weighing 280 ± 40 g following the recommendations of the Institutional Animal Research Committee according to international conventions on animal experimentation. After killing the animals, the thoracic aorta was quickly removed and cut into two rings of 3-mm length taking care not to damage the endothelial layer. The aortic segments were suspended in Krebs–Henseleit solution containing (mmol l–1): NaCl 115, KCl 4.6, KH2PO4 1.2, MgSO4 1.2, CaCl2 2.5, NaHCO3 25, glucose 11.1 and EDTA 0.02; pH 7.4. The organ bath containing 5 ml of the solution was gassed with 95% O2–5% CO2. The aortic segments were connected to isometric force displacement transducers coupled to a computer system (Power Laboratory 400, AD Instruments, Casterhill, NSW, Australia). The segments were considered ‘resting’ at the previously determined optimal resting force of 2 g, as determined by repeated exposure to 20 mmol l–1 KCl.
Hypoxia was induced by bubbling Krebs–Henseleit solution with 95% N2–5% CO2 for 20 min obtaining a PO2 of < 5 mmHg determined by a Clark-type electrode (Doval et al. 1998). In the hypoxic preconditioning model, the aortic rings were incubated for 1 min in Krebs solution gassed with 95%N2–5% CO2 followed by incubation for 5 min in Krebs solution gassed with 95% O2–5% CO2. This process was performed three times before incubating the aortic rings in the abovementioned hypoxic Krebs solution for 20 min. At the end of the ischaemic manoeuvre, the aortic rings were incubated in Krebs solution gassed with 95% O2–5% CO2 for 20 min (reoxygenation period). The concentration of the different gases was continuously monitored by a precalibrated multigas analyser (Datex Capnomac, Helsinki, Finland).
Endothelium-dependent relaxation to acetylcholine (Ach) (10–9–10–4 mol l–1) and endothelium-independent relaxation to sodium nitroprusside (SNP) (10–9–10–6 mol l–1) were tested on arteries precontracted with phenylephrine (10–5 mol l–1; Marques et al. 2001). The dose–response curves were determined in a cumulative manner. As previously described, all the experiments were performed in the presence of indomethacin (10–5 mol l–1) to block the effect mediated by the activation of cyclooxygenase (Lippolis et al. 2003; Oltman et al. 2003). To analyse the involvement of hsp90, further vasorelaxation studies were performed in the presence of geldanamycin (10 µg ml–1). Geldanamycin is an ansamycin antibiotic that binds to the ATP binding site of hsp90. Geldanamycin was added to the aortic rings just after the aortic rings were precontrated with phenylephrine. Moreover, the effect of HR and hypoxic preconditioning HR on the contractile response to phenylephrine (10–9–10–5 mol l–1) was also examined. The dose–response curves to phenylephrine were also determined in a cumulative manner.
The L-arginine competitor L-nitroarginine-methylester (L-NAME) 10–4 mol l–1 was added to the aortic rings to analyse the involvement of eNOS in the vasorelaxation responses (Tsuchida et al. 2000; Mital et al. 2002; Yakubu et al. 2004).
Lysis of aortic segments
To prepare lysates of the aortic segments, the aortic rings were pulverized with a homogenizer (ULTRA-TURRAX T8, Afora, Madrid, Spain) in hypotonic solution containing (mol l–1): Tris HCl 25 (pH 7.9), EDTA 0.5 and phenylmethylsulfonyl fluoride (PMSF) 1. The homogenates were then centrifuged at 12 000 g for 30 min at 4°C. The supernatants were removed and resuspended with 10% glycerol. Finally, they were frozen at –80°C until use.
Determination of eNOS–hsp90 interaction
Protein-A-agarose (10 µl; Affinity Research Products Ltd, Exeter, UK) was added to the lysates (50 µg total protein per sample) from the aortic segments and kept for 30 min at 4°C. The supernatants were obtained by centrifugation at 615 g for 5 min at 4°C and 20 µg total protein was incubated in the presence of anti-eNOS polyclonal antibody (Mouse IgG1, Clone 3 Transduction Laboratories, Lexington, UK) for 1 h at 4°C. Protein-A-agarose (20 µl) was then added to the supernatants which were incubated overnight at 4°C. Immunoprecipitates were collected by centrifugation at 615 g for 10 min at 4°C. The pellet was washed twice with 200 µl immunoprecipitation buffer containing (mmol l–1): Tris HCl 10 (pH 7.5), NaCl 140, and Nonidet-40 0.1%, leupeptine 21 µl ml–1, pepstatine 14.5 µl ml–1 and PMSF 10 µl ml–1, and then resuspended in 20 µl Laemmli buffer (Laemmli, 1970). The proteins were separated in 10% SDS-PAGE. The amount of eNOS protein that formed complexes with hsp90 was detected by immunoblotting using an anti-hsp90 antibody (1: 1000, mouse IgG1 Clone 68 Transduction Laboratories). The bands were visualized using the appropiate horseradish peroxidase (HRP)-linked secondary antibody and enhanced chemoluminescence (ECL) reagent and evaluated by densitometry (Courtois, 2003). The data were calculated as percentage of the control value. Prestained protein markers (Sigma) were used for molecular mass determinations.
Determination of serine1177-phosphorylated eNOS
The amount of serine1177-phosphorylated eNOS in the aortic segments was analysed by Western blot as mentioned above. In brief, the aortic rings were pulverized and solubilized in Laemmli buffer containing 2-mercaptoethanol. The proteins obtained were separated in denaturing 10% SDS-polyacrylamide gels. Equal amounts of proteins (20 µg per lane) estimated by bicinchonic acid reagent (Pierce, Rockford, IL, USA) were loaded. The proteins were then blotted into nitrocellulose (Immobillon-P, Millipore Ibérica, INC) and developed with a polyclonal anti-phospho-eNOS produced by immunizing rabbits with a synthetic phosphopeptide corresponding to residues surrounding serine1177 of human eNOS (Ser1177 eNOS, 1: 2000, Cell Signalling Technology). The bands were visualized and evaluated as mentioned above.
Detection of the content of superoxide anion and peroxynitrite in aortic segments
We detected the generation of superoxide anion by confocal microscopy. The oxidative fluorescent dye dihydroethidium (DHE, 5 µmol l–1) was used to evaluate in situ production of superoxide. DHE is freely permeable to cells and in the presence of superoxide anion is oxidized to ethidium, where it is trapped by intercalating with DNA (Rothe & Valet, 1990). Ethidium is excited at 488 nm with an emission spectrum at 610 nm. For peroxynitrite detection, 4,5-diaminofluorescein diacetate (DAF-2 DA, Sigma) was used as fluorescent probe. DAF-2DA has been used to detect endogenous NO (Pritchard et al. 2001). However, it has been recently shown that DAF-2DA detected peroxynitrite rather than NO (Roycowdhurry et al. 2002). DAF-2DA is excited at 492 nm with an emission spectrum at 515 nm.
For confocal analysis, aortic segments subjected to the abovementioned different experimental manoeuvres were frozen and cut into 30 µm-thick sections with a microtome and placed on a glass treated with 3-aminopropyltriethoxy-silane (APES) to enable the tissue to stick to the glass. DHE (2 µmol l–1) or DAF-2DA (10 µmol l–1) was added to each tissue section. The slices were incubated in a light-protected humidified chamber at 37°C for 30 min and then they were washed three times with PBS. Images were obtained with a Leica Confocal Microscope (Leica TCS SP2 Microsystems, Heidelberg, Germany). The microphotographs were then examined by three blinded observers who classified the fluorescence intensity using a numerical score: 0, very low intensity; 1, low intensity; 2, medium intensity; 3, high intensity; and 4, very high intensity.
Statistical analysis
Results are expressed as means ± S.E.M. Comparisons of the vasodilatation studies were performed by ANOVA. Bonferroni's correction for multiple comparisons was used to determine the level of significance of the P-value. To determine the statistical significance of the molecular biology parameters, Mann Whitney U test was used. P < 0.05 was considered as significant.
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Results |
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Relaxation in response to addition of Ach was significantly reduced in phenylephrine-precontracted aortic segments after HR compared with control aortic segments (Fig. 1A). IC50, which is a measure of sensitivity, was 5 ± 2.5 x 10–8 mol l–1 for control segments and 3 ± 1.2 x 10–7 mol l–1 for aortic segments subjected to HR (P < 0.05). A greater maximum response to Ach was also observed in control compared with HR-exposed aortic rings (Fig. 1A).
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There were no statistical differences between control, HR and hypoxic preconditioning in the vasoconstrictor response curves to increasing concentrations of phenylephrine (Fig. 2).
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After HR, the amount of hsp90 that coprecipitated with eNOS protein was higher than in control segments (Fig. 3A). Hypoxic preconditioning also enhanced the level of hsp90 associated with eNOS protein when compared with control aortic rings (Fig. 3A). There were no significant differences between HR and preconditioned aortic segments in the level of hsp90 that coprecipitated with eNOS protein (Fig. 3A). A similar pattern was observed when the homogenates were immunoprecipitated with the anti-hsp90 antibody and then the Western blot was developed with the anti-eNOS antibody (Fig. 3B).
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There were no differences in the amount of total eNOS protein and hsp90 expressed in control, HR and preconditioned aortic segments (Fig. 4). HR rat aortic segments showed a slight although significant increase in the content of serine1177-phosphorylated eNOS when compared with control aortic rings (Fig. 5A). Hypoxic preconditioning enhanced the level of serine1177 phosphorylation of eNOS protein in the vascular wall when compared with both control and HR aortic rings (Fig. 5A). The presence of geldanamycin (10 µg ml–1) reduced the content of serine1177 phosphorylation of eNOS protein in the vascular wall of hypoxic preconditioned aortic segments to level similar to that of HR segments (Fig. 5A). No differences were found in the expression of the constitutive protein ß-actin, supporting the specificity of the observed changes in the serine1177 phosphorylation of eNOS protein (Fig. 5A). The whole experiments were performed in tensioned aortic rings. Therefore we tested whether the resting force (2 g) affects hsp90 expression and serine1117 phosphorylation of eNOS. As shown in Fig. 5B, there were no differences in the level of expression of hsp90 between non-tensioned and tensioned aortic rings. However, the level of serine1177-phosphorylated eNOS was higher in tensioned than in non-tensioned aortic rings (Fig. 5B). We speculated that the tensioned vessel may try to generate a higher amount of NO following serine1117 phosphorylation of eNOS protein in an attempt to fight against the tension.
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Confocal microscopy experiments demonstrated greater superoxide anion content in the vascular wall of HR aortic segments compared with controls (Fig. 6A and C). Hypoxic preconditioning reduced the content of superoxide although it remained increased with respect to controls (Fig. 6A and C). Addition of geldanamycin (10 µg ml–1), an agent known to bind to the ATP binding site of hsp90 (Garcia-Cardena et al. 1998; Dimmeler et al. 1999), to preconditioned hypoxic aortic segments increased superoxide anion content in the vascular wall (Fig. 6A and C).
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Addition of 10–4 mol l–1 L-NAME reduced superoxide anion content in the vascular wall of HR aortic segments although it remained elevated with respect to control aortic segments incubated with L-NAME (Fig. 6A–C). L-NAME (10–4 mol l–1) also reduced superoxide anion content in geldanamycin-incubated aortic segments compared with that observed in geldanamycin-incubated aortic rings in the absence of L-NAME (Fig. 6A–C). L-NAME failed to significantly modify superoxide anion content in HR preconditionied aortic segments (Fig. 6A–C).
Confocal microscopy experiments also showed a greater DAF-2DA fluorescence in HR segments compared with controls, suggesting the presence of peroxynitrite (Fig. 7A and C). In hypoxic preconditioning segments, the DAF-2DA fluorescence signal was significantly reduced with respect to that of HR but remained increased with respect to control segments (Fig. 7A and C). Geldanamycin increased the fluorescence signal of DAF-2DA in the preconditioned segments (Fig. 7A and C). The presence of L-NAME (10–4 mol l–1) reduced peroxynitrite formation in HR, HR preconditioned and geldanamycin-incubated aortic segments to a level similar to that observed in L-NAME-incubated control (Fig. 7A–C).
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Discussion |
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We first observed that HR aortic segments show a reduced Ach-induced vasorelaxation. However, the same vessels retained the endothelium-independent vasorelaxing response to an exogenous NO-donor, SNP. These findings suggest that, under our experimental conditions, HR selectively altered the functionality of the endothelium, without affecting that of smooth muscle cells which is in accordance with previously reported studies in other experimental HR models, including an in vitro study in which hypoxia reperfusion was produced in isolated rat aortic segments (Yokoyama et al. 1996) similar to in the present study.
Hypoxic preconditioning improved endothelium-dependent vasodilatation in response to Ach, suggesting the existence of an endothelial adaptation that partially protects the endothelial functionality after HR.
Although Ach-induced vasorelaxation was impaired in both HR and preconditioned aortic segments compared with controls, the vasoconstrictor response to phenylephrine was not affected. Richard et al. (1994) have also reported no significant differences in the contractile response in rat coronary arteries isolated from hearts previously exposed to ischaemia–reperfusion and hypoxic preconditioning. Moreover, Yokoyama et al. (1996), using an in vitro model of vascular hypoxia similar to the one used by us, have also reported a preserved vasoconstrictor response to phenylephrine. A similar observation has been reported in isolated aortic segments from stroke-prone hypertensive rats and eNOS knockout mice (Brandes et al. 2000; López-Farré et al. 2002). The fact that the vasoconstrictor response in the hypoxic segments remained preserved could mean that other vasodilating systems may replace the impaired NO-dependent vasorelaxation produced in the vascular wall damaged by the hypoxia–reperfusion processes, thus counterbalancing the contractile response.
The importance of the endothelium in the protection of ischaemic organs such as the heart and kidney has been previously demonstrated. As an example, permanent damage to peritubular capillaries has been observed in rats subjected to renal ischaemia which was associated with a predisposition to the development of renal fibrosis (Basile et al. 2001). Moreover, implantation of endothelial cells or of eNOS protein in the renal microvasculature results in an important functional protection of the ischaemic kidney, an effect that was also achieved by treatment with a spontaneous NO releaser (Matsumura et al. 1998; Brodsky et al. 2002). A protective effect of the endothelium and NO has been also observed in ischaemic myocardium (Jugdutt, 2002). Taken together, these observations suggest the importance of the endothelium, eNOS protein and NO in the protection against the ischaemic injury.
eNOS activity is stimulated by the interaction of eNOS with hsp90 which facilitates the Akt protein kinase B (Akt)-dependent phosphorylation of serine1177 on eNOS protein (Garcia-Cardena et al. 1998; Dimmeler et al. 1999). We thus determined the level of interaction between eNOS protein and hsp90 in both HR and preconditioned aortic segments.
The complex formed between hsp90 and eNOS protein was increased in both HR and preconditioned aortic segments compared with controls. However, the content of serine1177 phosphorylated eNOS in HR preconditioned segments was of greater magnitude than in HR. This seems paradoxical as the level of interaction between hsp90 and eNOS was similar in HR and preconditioned segments.
Emerging evidence suggests that under pathological conditions eNOS may also generate superoxide anion (Pritchard et al. 2001). In our study, the amount of both superoxide anion and peroxynitrite in the vascular wall was increased in the HR vessels and was partially reduced by hypoxic preconditioning. Moreover, the presence of geldanamycin (an ansamycin antibiotic that binds to the ATP binding site of hsp90 thereby inhibiting the ATP–ADP cycle used for the interaction with client proteins such as eNOS) increased the amount of superoxide anion and peroxynitrite in the HR preconditioned aortic segments and was associated with a reduction in the level of serine1177-phosphorylated eNOS protein. Geldanamycin contains a quinone group and such molecules are known to have redox-active properties (Bachur et al. 1978). Indeed, it has been shown that treatment of endothelial cells with geldanamycin may increase superoxide anion generation independently of eNOS activity by a non-L-NAME-inhibitable mechanism (Dikalov et al. 2002). In this regard, it has been also demonstrated that, a substrate analogue inhibitor that blocks eNOS activity at the L-arginine domain, prevents both NO and superoxide anion formation by eNOS (Pritchard et al. 2001; Shi et al. 2002). In our study, the finding that L-NAME diminished the content of superoxide anion in both HR and geldanamycin-incubated hypoxic preconditioned aortic segments supports the involvement of eNOS in superoxide anion formation within the vascular wall and discards a putative production of superoxide anion by the quinone group of geldanamycin. Taken together, our results then suggest that the conformation of hsp90 that interacts with eNOS protein, i.e ATP– or ADP–hsp90 conformation, may play an important role in the level of serine1177 phosphorylation of eNOS protein and in mediating the balance of NO and superoxide anion generation. Accordingly, Pritchard et al. (2001) have shown that the generation of superoxide anion was favoured with respect to NO in geldanamycin-incubated cultured coronary endothelial cells.
The functional experiments in geldanamycin-incubated aortic segments are difficult to interpret. Geldanamycin reduced Ach-dependent vasorelaxation in control, HR and hypoxic preconditioned aortic segments. Moreover, the degree of attenuation of the Ach-dependent vasorelaxing response by geldanamycin was similar in HR and hypoxic preconditioned aortic segments. However, Ach-induced vasorelaxation remained preserved in geldanamycin-incubated hypoxic preconditioned aortic segments with respect to geldanamycin-incubated HR aortic segments, suggesting that endothelial hypoxic preconditioning persisted in the presence of geldanamycin. Taken together, these findings may suggest that the serine1177 phosphorylation of eNOS protein and the conformation of the hsp90–eNOS interaction are important to attenuate the endothelial dysfunctionality induced by HR but are probably not the mechanisms by which functional endothelial hypoxic preconditioning occurs.
To our knowledge, this work demonstrates for the first time the induction of endothelial hypoxic preconditioning, studying the whole vascular wall in vitro. However, the concept of endothelial hypoxic preconditioning has been previously raised by others in experiments performed in cultured endothelial cells (Zhou et al. 1996; Zahler et al. 2000; Bae et al. 2003; Thors et al. 2003).
We must also point out that, although the endothelium-dependent vasodilatation was improved by hypoxic preconditioning, it remained impaired compared with that of control aortic segments. This fact suggests that endothelial hypoxic preconditioning, at least in our in vitro model, could not preserve the whole endothelial functionality. Accordingly, in the presence of L-NAME, the content of superoxide anion in the vascular wall of both HR and hypoxic preconditioned aortic segments remained elevated with respect to the controls, suggesting that other mechanisms, in addition to eNOS, are a source of superoxide anion under hypoxic conditions.
The potential pathophysiological relevance of our findings needs to be specifically addressed in in vivo conditions. In this regard, a limitation of our study may be that the experiments were performed using aortic segments instead of smaller arterial vessels such as renal and coronary arteries. However, renal and coronary endothelial cells also generate NO and express eNOS protein as does the aortic endothelium (Heeringa et al. 2001; Ishimura et al. 2002; Hambrecht et al. 2003). In this regard, it has been recently shown that elevated eNOS expression after reperfusion in living related-donor renal transplantation enhanced the recovery from renal ischaemia and reduced late graft deterioration (Ishimura et al. 2002). Therefore, the existence of endothelial adaptation to HR injury may be important in renal and myocardial protection against HR injury. Another limitation of our study is that only one endothelium-dependent vasodilator was used (Ach). Therefore, we have to be cautious because the impairment observed in the Ach-dependent vasorelaxating response in HR segments may reflect a selective defect for the muscarinic receptors linking to the NO system. Further studies are then warranted.
In conclusion, endothelial hypoxic preconditioning could be induced in isolated aortic segments by short periods of hypoxia. However, the fact that endothelial hypoxic preconditioning persisted in the presence of the hsp90–ATP binding inhibitor, geldanamycin, suggests that other molecular mechanisms than the stimulation of serine1177 phosphorylation of eNOS protein are involved in the endothelial adaptation to HR.
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), HR (
) and hypoxic preconditioned (
) isolated rat aortic segments. B, vascular relaxation induced by sodium nitroprusside (SNP) in control (
) and hypoxic preconditioned (